PL48: mechanism of a new cell growth regulatory gene that inhibits proliferation and promotes differentiation in human cytotrophoblast

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Morrish This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8584838/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: PL48 is a novel human placental differentiation-associated gene that is widely expressed in normal tissues. Methods: To determine the effect and mechanism of PL48 on cell proliferation, apoptosis, and cytotrophoblast differentiation, several cell models were used. Results: Antisense PL48 transfection into term human trophoblast reduced spontaneous cytotrophoblast differentiation into syncytium by 19.9 + 1.1 %. Transiently transfected MCF-7 cells demonstrated a 19.5-25.7% decrease in 3 H-thymidine uptake concomitantly with a 20% decrease in cell number and 3-fold increase in apoptotic cells shown by TUNEL labeling. Stable retroviral transfection of PL48 into MCF-7 cells resulted in a 50% decrease in 3 H-thymidine uptake after gene promoter induction. Estrogen-induced proliferation of MCF-7 cells was not affected by PL48 induction with or without tamoxifen, indicating PL48 does not act through the estrogen receptor. Transfected MDA-MB231 cells showed a 20% decrease in 3 H-thymidine uptake but no induction of apoptosis indicating that a functional p53 is required for PL48-induced apoptosis. Stably transfected MCF-7 cells showed an upregulation of p53 and p21 mRNA. Peroxynitrite, but none of xanthine/xanthine oxidase, hypoxia, or cytokines (EGF, IFNγ, TNFα, TGFβ) induced PL48 in trophoblast cells.10cGy gamma irradiation did not induce PL48 in MCF-7 cells. Discussion: We conclude that PL48 has multiple actions including inhibition of proliferation independently of p53, induction of apoptosis through the p53 pathway, and enhancing differentiation in cytotrophoblast. PL48 p53 apoptosis placenta differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction A key step in the development of the placenta is the formation of the syncytium from several precursor types of cytotrophoblast cells (Liu 2018;Lawless 2023). In human placenta, multiple factors and cell signaling pathways regulate cytotrophoblast fusion into syncytium (Lawless 2023; Io 2021; Xiao 2020; Castel 2022 ; Knofler 20219) and can be mimicked in vitro by cell reprogramming and regulation of stem cells in vitro (Castel 2022 ). Activation of apoptosis (caspase 8) is also required for cytotrophoblast differentiation (Huppertz 2001; Black 2004). In support of this concept, we found that the cytokine PL74 (MIC-1) had a bifunctional capacity to induce both differentiation and apoptosis, suggesting such factors may act as switches between differentiation and apoptosis (Li 2005). In a search for additional regulators of trophoblast differentiation, we cloned the novel gene PL48 from a subtractive library of in vitro differentiating normal human cytotrophoblast cells (Morrish et al 1996 ; Dakour et al 1997 ). These cells spontaneously exit the cell cycle and differentiate into a syncytium and are a model of spontaneously differentiating trophoblast Morrish et al 1997 ). PL48 has no similarities to known functional gene classes and initial studies demonstrated that it had low or absent expression in 6 different tumor cell types (Dakour et al 1997 ). PL48 has also been found as part of genome cloning projects in man and mouse and is localized to chromosome 6 (Jea Kawai 2002 ). Because of the association of PL48 with growth arrest and differentiation, we wished to directly determine its interaction with p53 and the resultant outcome on cell proliferation, apoptosis, and differentiation in several cell models including normal human term trophoblast, MCF-7 and MDA-MB231 breast cancer cells, which have wild type p53 and mutated nonfunctional p53 respectively (Bartek 1990). Cells exiting the cell cycle may undergo various fates including entering a resting state, differentiation, or cell death. A key switch in this process is p53. Multiple environmental stress signals including hypoxia, DNA damage, drugs, serum deprivation, UV and gamma radiation, aberrant oncogene expression and oxidative stress can stabilize p53 protein expression leading to growth arrest and/or apoptosis (Cox 1997 ; Fridman 2003 ; Ashcroft 2000; Oren 2003 ; Oren et al 2002; Ueda 2002; Fritz 2003). It is not yet clearly understood how p53, once activated, can direct a cell to one of many fates including transient or permanent growth arrest, accelerated DNA repair, terminal differentiation, or apoptosis (Fridman 2003 ; Oren 2003 ; Oren, Damalas 2002). There are a large number of downstream target genes of p53 including the cell cycle regulator p21 Cip1 , and both induced and repressed genes are required for apoptosis (Weber 2003 ). Furthermore, there can be both p53-dependent and independent inhibition of the cell-cycle (Kastan 2004 ). Aberrant control of these growth processes may lead to cancer and because of its pivotal position, p53 is frequently found to be mutated in cancers. In these studies, we show that PL48 has both p53-dependent and independent modes of action in regulation of cell cycle arrest and apoptosis. Materials and Methods Tissue Distribution and Subcellular Localization The study has received approval of the institutional ethics board. A normal human tissue dot blot representing most human tissues was obtained from Clontech Laboratories Inc., Palo Alto, CA, and probed with a full-length PL48 cDNA probe (see below). Subcellular localization of PL48 in normal first trimester and term placenta samples was performed by in situ hybridization. First trimester and term normal placental tissue were obtained from therapeutic terminations of pregnancy and normal deliveries, respectively, and snap frozen in liquid nitrogen and stored at -80 o C until use. In situ hybridization was performed by first fixing small pieces of frozen placenta or breast cancer tissue in 4% paraformaldehyde in active DEPC overnight, followed by 30% sucrose overnight. 5–10 µM sections were cut onto Super-frost slides (Fisher), air dried and stored at -80 o C until use. For hybridization, slides were treated with proteinase K for 30 min at room temperature, postfixed with 4% paraformaldehyde in DEPC-PBS for 10 min, treated with triethanolamine-acetic anhydride for 10 min, washed in PBS-DEPC, and equilibrated in 5xSSC for 10 min. Slides were prehybridized for 2 h at 65 o C in 50% formamide, 25% 20xSSC and salmon testes DNA (Invitrogen). The PL48 cDNA probes were constructed by PCR using the sense primer GAA TTT AAT ACG ACT CAC TAT AGG GCG AGC TGG CTT TGC ACG CCT CT (T7 promoter underlined) or the antisense primer GAA TTG GAT TTA GGT GAC ACT ATA GAA TAC GCT GGT GTC ATC CAT ACC CTC AT (SP6 promoter underlined). DNA products were purified on 1% agarose gels, electroeluted and purified with QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer’s instructions. Probes were denatured at 80 o C for 5 min then labeled with digoxigenin using a kit (Roche) according to the manufacturer’s instructions. Probes were applied in prehybridization solution to the slides using a Mound Hybridization Sealing System (Molecular Probes) overnight at 65 o C. After hybridization, slides were washed with 2xSSC for 30 min at 37 o C, 2xSSC for 60 min at 65 o C, then 0.1xSSC for 60 min at 65 o C. Antibody solution (1:2000) for digoxigenin was added for 2 h at 37 o C, the slides washed, then color developed with NBT/BCIP solution at 37 o C. Slides were dehydrated in increasing 35–100% ethanol solutions, counterstained with Vector Nuclear Fast Red (Vector Lab), and photographed. Semi-quantitative PCR was performed to compare first trimester and term placental expression of PL48. The same protocol as above was used and primers for PL48 were as above. The annealing temperature used was 60 o C. An internal standard using GAPDH was used as amplification control. Primers for GAPDH were: sense: 51-GAA GGT GAA GGT CGG AGT C-31; antisense: 51-GAA GAT GGT GAT GGG ATT TC − 31. Reverse transcription was performed using the Superscript II Reverse Transcription Reagents kit (GIBCO BRL) according to the manufacturer’s instructions. The resultant cDNA mixture was stored at -20 o C until ready for PCR. As a negative control, the reaction was also performed in the absence of reverse transcriptase in randomly chosen samples. The PCR reaction was performed using the Tag DNA Polymerase kit (GIBCO BRL). The PCR mixture was initially denatured at 95 o C for 10 min, followed by 40 cycles of 95 o C for 30 s, 60 o C for 60 s, and 72 o C for 30 s, with an extension cycle at 72 o C for 10 min. The PCR products along with a nucleotide ladder were run on a 2% agarose gel treated with ethidium bromide and photographed under ultraviolet light. Cell culture Because term human cytotrophoblast cells do not proliferate in vitro (Morrish et al 1987 ), and existing first trimester cytotrophoblast cell lines (HTR8/SVneo and SGHPL-4) have blocked p53 due to their creation using large T antigen (Aboagye-Mathiesen 1996), we chose to use breast cancer cell lines in which wild-type p53 estrogen-receptor positive MCF-7, and estrogen-receptor negative p53-mutated MDA-MB231 breast cancer cells (obtained from the American Type Culture Collection, Rockville, MD) were available and therefore could be used to determine p53-dependent and p53-independent effects. These cells were grown in 24 well multiwell plates (Corning, New York) in phenol red-free DMEM (Sigma Chemical Co., St. Louis, MO) containing 10% charcoal-stripped fetal calf serum containing 50 µg/ml penicillin-streptomycin (Sigma). Term and first trimester placentas were obtained from normal deliveries and therapeutic terminations of pregnancy. Tissue for in situ hybridization was snap frozen in liquid nitrogen and stored at -70 o C. Term trophoblast cultures were performed as previously described using a trypsin-DNAse I digestion that produces a cell preparation over 95% pure for cytotrophoblast with fewer than 5 vimentin-positive cells per 10 5 cells and which is essentially free of syncytial fragments and identical in purity and function to CD9/HLA class I/II immunopurified cells (Morrish et al 1987 ; Guilbert 2002; Dakour et al 1999 ). Cells were plated at 6–8 x 10 6 cells/dish in 100 mm Petrie dishes (Corning) for mRNA studies and cultured in 10% FBS- DMEM-penicillin-streptomycin as described (Guilbert 2002). Cells were attached for 2 h, then the medium changed to serum-free DMEM. These cells do not proliferate but rapidly differentiate spontaneously over 18–24 h toward a syncytial phenotype including upregulation of most syncytial gene products and formation of morphological syncytium, but have a villous cytotrophoblast phenotype in early ( ~ < 3 h) culture (Morrish et al 1997 ; Dakour et al 1999 ; Garcia-Lloret 1996). Induction of PL48 Expression To determine inducers of PL48 expression, MCF-7 and normal trophoblast cells were used. Term trophoblast cultures were exposed to 18 uM peroxynitrite or 5 µ units/ml xanthine oxidase/100 µM xanthine for 18 h to induce an oxidative stress, or 2% oxygen for 18 h using an hypoxia incubator (Biospherix PRO-OX model 110 regulator and chamber) to induce an hypoxic stress. DNA damage was induced by exposing MCF-7 cells for 18 h to 10 cGy gamma radiation. Cytokine effects were determined by adding 10 ng/ml EGF, 10 ng/ml TGFβ1, 10 ng/ml TNFα or 100 U/ml IFNγ (Upstate) or controls to culture medium of term cytotrophoblast cultures for 24 h. Four separate experiments were performed for each of hypoxia, peroxynitrite and xanthine/xanthine oxidase, and two experiments for cytokine effects. Transfections A full length insert of PL48 as previously reported (Dakour et al 1997 ) was cloned into pcDNA3 for transient transfections of MCF-7 and MDA-MB231 cells. Transient transfections were performed using Lipofectin by adding 0.5 µg/ml pcDNA3-PL48 or 0.5 µg/ml pcDNA3 alone (control) DNA to cells. Three days after transfection, cells were evaluated comparing control untransfected cells, empty vector alone and vector-PL48 as follows: (1) cell counts using a hemocytometer, (2) 3 H-thymidine uptake, and (3) apoptosis quantitation using the TUNEL method. Three separate transient transfections of MCF-7 and two of MDA-MB231 cells were evaluated. Transfection efficiency was determined in MCF-7 and HL-60 cells (see below) by co-transfecting the β-galactosidase gene in pCMV, staining transfected cells with X-Gal, and counting blue-stained (positively-transfected) cells (In Situ β-Galactosidase Staining Kit, Stratagene Cloning Systems, La Jolla, CA). Stable transfectants of PL48 with an ecdysone-inducible promoter were prepared according to the manufacturer’s instructions (Ecdysone-Inducible Expression Kit, In Vitrogen, San Diego, CA). In brief, PL48 was cloned into the multiple cloning site of pIND and the resulting construct co-transfected with the pVgRXR vector into MCF-7 cells. Transfected cells were selected by using double selection with ampicillin and Zeocin as indicated by the manufacturer. The PL48-transfected cells were assessed for induction of PL48 by adding 1 µM of the ecdysone analogue muristerone A to the medium for 24 hours and then harvesting the cells for mRNA and northern blot analysis as described below. 3 H-thymidine uptake was performed on uninduced cells and muristerone A-induced cells. We also studied induced and uninduced cells to which 10 − 6 M tamoxifen or 10 − 9 M estradiol had been added to determine if estrogen or estrogen-receptor mediated events could induce PL48 in MCF-7 cells. Stable transfectants were also constructed in MCF-7 and MDA-MB231 cells using a retroviral vector (Clontech, Tet Off), according to the manufacturer’s instructions. Induction of the promoter was achieved by removing tetracycline from the culture medium. Effects of PL48 on Human Cytotrophoblast Differentiation To determine the effects of PL48 on human cytotrophoblast differentiation, a full-length antisense construct of PL48 was prepared in pcDNA3. The antisense construct or empty vector control was transiently transfected as above into term primary human cytotrophoblast cultures (prepared by CD9/HLA class I/II immunopurification) immediately after attachment of cells. Transfection was allowed to proceed for 72 h at which time the cultures were stopped and fixed for immunostaining for desmoplakin. Desmoplakin staining was performed as previously described (Douglas 1990 ). Formation of syncytia was quantitated by counting nuclei within desmoplakin-stained outlines (Douglas 1990 ). The ratio of multinucleated cell groups (at least two nuclei) to total number of nuclei was calculated as a measure of syncytial unit formation and expressed as a percentage. Two separate transfection experiments were performed immediately after thawing and reconstituting two different cell preparations. Seven randomly chosen microscope fields were counted in total to determine percent syncytial unit formation, and 78–125 nuclei per field were counted in each experiment. 3 H-thymidine uptake 1 µCi 3 H-thymidine was added for 3 h to quadruplicate wells of MCF-7 or MDA-MB231 cells. The cells were then washed three times with PBS, briefly trypsinized with 0.25% trypsin, and cells centrifuged for 3 min at 300 x g. The cell pellet was dissolved in liquid scintillation cocktail (EcoLite, ICN Biochemicals, Costa Mesa, CA) for liquid scintillation counting. TUNEL assay of DNA nicking (apoptosis) This assay detects nuclear DNA fragmentation in apoptotic cells using terminal deoxynucleotidyl transferase (TdT)-mediated DTU)-biotin DNA-nick end labeling of free 3'-OH termini and was performed using a kit (In Situ Death Detection Kit, AP, Boehringer Mannheim Canada, Laval, PQ) following the manufacturer’s instructions. Three separate microscope fields containing at least 150 cells of each of control or PL48-transfected MCF-7 or MDA-MB231 cells were counted for TUNEL-positive cells. Northern blot analysis mRNA was extracted by use of RNeasy Mini Kit (QIAGEN Inc., Mississauga, ON). Fifteen micrograms of the extract were separated on a 1.2% agar-formaldehyde denaturing gel and transferred to Nytran Plus membrane (Schleicher & Schuell, Keene, NH) by standard methods (Sambrook 1989). The blot was probed with a full length PL48 cDNA probe labeled by random priming labeling using a DNA Labeling System (GIBCO BRL Life Technologies, Gaithersburg, MD) as previously described (Morrish, Linetsky 1996). RNA loading was quantitated by reprobing the blots with 18 S rRNA and performing densitometry. Results Tissue distribution and Localization of PL48 All normal human tissues tested expressed PL48mRNA (Fig 1). In situ hybridization of PL48 in placenta showed expression exclusively in syncytiotrophoblast in a nuclear and perinuclear distribution (Figs 2,3). Expression was quite weak in the first trimester (Fig 2) but strong at term (Fig 3), indicating gestational dependence of expression. PL48 expression by semi-quantitative PCR confirms this gestational difference (Figs 4a,4b). Function and signaling pathway of PL48 We transiently transfected PL48 into MCF-7 and MDA-MB231 cells and measured 3 H-thymidine uptake, cell number, and apoptosis using TUNEL (Table 1). 3 H-thymidine uptake decreased significantly ( 3 H-thymidine uptake: MCF-7: p<0.05; MDA-MB231: p<0.05). Cell number also decreased significantly. Quantitation of apoptotic cells using TUNEL showed a significant increase after PL48 transfection into MCF-7 cells (control: 5.1 + 0.2%; PL48: 16.0 + 1.4%; t-test, p < 0.025; Fig 5) but no induction of apoptosis after transfection into MDA-MB231 cells (control 5.5 + 1.0%, PL48 5.0 + 1.1% p = NS; Fig 5). Transfection efficiency as determined by co-transfection of galactosidase was 15-20%. Initial attempts to stably transfect PL48 using pcDNA3 were unsuccessful because PL48 expression was lethal. We therefore made stable transfectants in MCF-7 cells using an ecdysone-inducible promoter system. In this preparation, PL48 expression was significantly induced using the ecdysone analogue muristerone A. Wild-type cells expressed very low or absent levels of PL48 not detectable on northern blot (Fig 6). Reprobing the same blot with p53 and p21 demonstrated that both genes were strongly induced in MCF-7 transfected cells compared to untransfected cells. We also stably transfected MCF-7 and MDA-MB231 cells with a retroviral vector with an inducible promoter (Clontech, Tet Off). After promoter induction, PL48 induced a 37-63% decrease in 3 H-thymidine uptake in these cells (Table 2). Presumably because of incomplete suppression of promoter activity, transfected cells with uninduced promoter showed 17-44% reduction in 3 H-thymidine uptake. To determine if PL48 acted through the estrogen receptor, ecdysone-inducible stably transfected MCF-7 cells were exposed to 10 -9 M estradiol or 10 -6 M tamoxifen, both with and without muristerone A induction and compared to control untransfected cells (Table 2). Induced cells had approximately 20% reduction in 3 H-thymidine uptake, similar in magnitude to the effect of tamoxifen during this exposure period. Estradiol induced a large increase in 3 H-thymidine uptake, but concomitant PL48 expression still resulted in about a 20% reduction of the growth-stimulatory effect of estradiol. Induction of PL48 Expression To determine if DNA damage were an inducer of PL48, MCF-7 cells were exposed to 10 cGy of gamma irradiation for up to 18 h (Fig 7, top panel). No induction was observed. We also tested the effects of 2% hypoxia and oxidative stress (induced by peroxynitrite and xanthine/xanthine oxidase) in term trophoblast (Fig 7, middle panel). Severe oxidative stress as induced by peroxynitrite, but not weaker oxidative stress induced by xanthine/xanthine oxidase nor hypoxia, induced PL48. Previously, we have shown that the non-physiologic agent DMSO will also induce PL48 in HL-60 cells, but this is presumed to be a concomitant of differentiation and not a direct inducer of PL48 (Morrish et al 1991). Exposure of trophoblast cells to EGF, TNFα, TGFβ, or INFγ had no effect on PL48 expression (Fig 7, bottom panel). Effects of PL48 on Cytotrophoblast Differentiation Transfection of antisense PL48 reduced spontaneous cytotrophoblast differentiation into syncytial units by 19.9+1.1% (p<0.05, ANOVA) (Fig 8). Discussion We first determined the expression and localization of PL48 in human tissues, and found that PL48 was widely distributed in all normal tissues tested (Fig. 1 ). In situ hybridization and semi-quantitative PCR demonstrated that placental expression was restricted to syncytium, with a gestational dependence in which term syncytium showed higher expression compared to first trimester (Figs. 2 , 3 ). Because primary term human cytotrophoblast cells do not proliferate, and first trimester cytotrophoblast cell lines (eg HTR8/SVneo) have mutated p53, we elected to study effects of PL48 on cell proliferation and apoptosis using two breast cancer cell lines, MCF-7 and MDA-MB231, as model systems of wild-type and mutated p53 respectively, We found that PL48 overexpression by transfection significantly inhibited cell proliferation regardless of p53 status, up to 63% when using a retroviral vector. During chronic exposure in stably transfected cell lines, we also found that PL48 displayed a lethal phenotype when highly expressed, requiring the use of suppressible promoter systems to allow generation of stable transfectants. The inhibition of growth in MCF-7 cells was not through the estrogen receptor because blocking of the receptor with tamoxifen did not prevent proliferation and the effect occurred in MDA-MB231 cells which lack the estrogen receptor. To investigate further the mechanism of reduction in cell growth in MCF-7 cells, we assessed the effects of PL48 on apoptosis (Fig. 5 ). These data demonstrated that PL48 significantly induced apoptosis in MCF-7 cells but not in MDA-MB231 cells. Since MCF-7 cells contain a wild-type p53 but MDA-MB231 cells have a nonfunctional mutated p53 (Fridman 2003 ), the data indicate PL48 requires functional p53 to induce apoptosis. Stable overexpression of PL48 in MCF-7 cells resulted in induction of p53 and p21 mRNA (Fig. 6 ). P53 transcriptionally activates p21, thus leading to cell cycle arrest (Cox 1997 ). Thus, PL48 can act through this pathway to inhibit cell proliferation. However, it is known that p21 expression can also be induced independently of p53 by a variety of factors including MyoD, NGF, oxygen radicals and TGFβ (Cox 1997 ). Since PL48 also caused growth inhibition in MDA-MB231 cells which lack a functional p53 and are unable to induce p21, the data would indicate PL48 is also able to bypass p53 by an alternate pathway (Oren 2003 ; Huo 2004 ; Chen 1996) to directly induce p21 and cell cycle arrest. P53 can mediate apoptosis signals (Waldman 1996) and this can occur in cells lacking p21 (Polyak 1996; Agarwal 1998), but the precise pathway or mechanism of this effect is unknown (Cox 1997 ; Chen 1996). In the model systems we used, however, PL48 appears to require p53 to cause apoptosis, since MDA-MB231 cells do not undergo this process when PL48 is overexpressed. Several genotoxic stresses are known to induce p53 resulting in apoptosis (Ashcroft 2000; Ueda 2002; Fritz 2003; Weber 2003 ; Agarwal 1998). To determine if any of these factors induced PL48, we tested gamma irradiation on MCF-7 cells, and 2% hypoxia, cytokines (TNFα, TGFβ, and IFNγ) and oxidative stress on trophoblast cells (Fig. 7 ). Of these, only strong oxidative stress (peroxynitrite) induced PL48 (Fig. 7 ). TNFα and IFNγ are known to induce apoptosis in trophoblast cells (Douglas 1990 ) but such actions would not appear to involve PL48 induction (Fig. 7 ). Since PL48 expression is coincident with cytotrophoblast differentiation into syncytium, we tested whether PL48 expression could mediate this differentiation process. We found that antisense PL48 transfection (Fig. 8 ) significantly inhibited spontaneous cytotrophoblast differentiation as assessed by the formation of desmoplakin-defined syncytial units. Since PL48 has high expression in placental syncytium, the results imply that PL48 may contribute to the induction and maintenance of the differentiated syncytial state. We have previously shown that EGF induces (Morrish et al 1987 ) and TGFβ1 inhibits (Morrish et al 1991 ) cytotrophoblast differentiation. However, neither of these cytokines altered PL48 expression, indicating their action in modulating differentiation was not through PL48 (Fig. 7 ). The actions of PL48 to regulate not only differentiation but also apoptosis and proliferation appear analogous to similar properties of the cytokine PL74 (MIC-1) as we have demonstrated (Li et al 2005 ). Huppertz et al have shown that human cytotrophoblast differentiation into syncytium are coupled, with inactive apoptotic proteins being present in cytotrophoblast, and these proteins then become activated with an obligatory activation of caspase 8 when the cells fuse to form syncytium (Huppertz 2001; Black 2004). In this context, cytokines and genes such as PL74 (MIC-1) and PL48 thus can regulate both apoptosis and differentiation and so may control the degree to which each process occurs (Li et al 2005 ). PL48 thus appears to be a new promoter of cytotrophoblast differentiation that also has capabilities to induce apoptosis or cell cycle arrest depending on whether wild-type or mutated p53 is present in a cell. It is also an upstream regulator of p53, possibly acting in response to severe cellular oxidative stresses. PL48 effects are particularly potent on growth inhibition. Whether the end result of PL48 action is apoptosis or only growth arrest appears to depend at least in part on the functional status of p53. Since the ultimate result of p53 activation (growth arrest, apoptosis, terminal differentiation) is thought to be dose-dependent on the degree of p53 activation (Oren 2003 ) this may therefore be a factor in determining which PL48 effects take place. As PL48 is expressed in many human tissues (Fig. 1 ) the data imply PL48 may have similar functions of growth inhibition, apoptosis and growth regulation in diverse tissues. Declarations Acknowledgements Supported by the Canadian Institutes of Health Research. We wish to thank Larry J Guilbert, PhD, Departments of Medicine and Medical Microbiology and Immunology for the CD9/HLA I/II purified cells and Hongshi Li, MSc, (current address Kinda Bioengineering, Yueyang, PR, China), Bonnie Winkler-Lowen BSc, Dept. of Medical Microbiology and Immunology, and Xin Fang, PhD, Perinatal Research Centre, University of Alberta,, for technical assistance. Contributors: I declare that I participated in the design and conduct of the study. J Dakour conducted most of the studies. DW Morrish designed the study and conducted some of the work. There is no conflict of interest by either author. Declaration of interests : none References Aboagye-Mathiesen G, Zdravkovic M, Toth ED, Graham CH, Lala PK, Ebbesen P (1996) Altered expression of the tumor suppressor/oncoprotein p53 in SV40 Tag-transformed human placental trophoblast and malignant trophoblast cell lines. 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Liu Y, Fan X, Wang R, Lu X, Dang Y-L, Wang H, Lin H-Y, Zhu C, Ge H, Cross JC, Wang H (2018) Single-cell RNA-seq reveals the diversity of trophoblast subtypes and patterns of differentiation in the human placenta. Cell Research, 28:819-832. https://doi.org/10.1038/s41422-018-0066-y Morrish DW, Bhardwaj D, Dabbagh LK, Marusyk H, Siy O (1987) Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J Clin Endocrinol Metab 65:1282-90. Morrish DW, Bhardwaj D, Paras MT (1991) Transforming growth factor b1 inhibits placental differentiation and human chorionic gonadotropin and human placental lactogen secretion. Endocrinology 129:22-26. Morrish DW, Dakour J, Li H, Xiao J, Miller R, Sherburne R, Berdan RC, Guilbert LJ (1997) In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation program that requires EGF for extensive development of syncytium. Placenta 18:577-85. Morrish DW, Linetsky E, Bhardwaj D, Li H, Dakour J, Marsh RG, Paterson MC, Godbout R (1996) Identification by subtractive hybridization of a spectrum of novel and unexpected genes associated with in vitro differentiation of human cytotrophoblast cells. Placenta 17:431-41. Oren M (2003) Decision making by p53: life, death and cancer. Cell Death Differ 10:431-42. Oren M, Damalas A, Gottlieb T, Michael D, Taplick J, Leal JFM, Maya R, Moas M, Seger R, Taya Y, Ben-Ze’ev A (2002) Regulation of p53. Intricate loops and delicate balances. Annals NY Acad Sci 973:374-383 Polyak K, Waldman T, He TC, Kinzler KW, Vogelstein B (1996) Genetic determinants of p53-induced apoptosis and growth arrest. Genes Dev 10:1945-52. Sambrook J, Fritsch EF , Maniatis T (1989) Extraction, purification, and analysis of messenger RNA from eukaryotic cells. Sambrook J, Fritsch EF, Maniatis T. Cold Spring Harbor Press Ueda S, Masutani H, Nakamura H, Tanaka T, Ueno M, Yodoi J (2002) Redox control of cell death. Antioxid Redox Signal 4:405-14 Waldman T, Lengauer C, Kinzler KW, Vogelstein B (1996) Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381:713-16. Weber JD, Zambetti GP (2003) Renewing the debate over the p53 apoptotic response. Cell Death Differ 10:409-12. Xiao Z, Yan L, Liang X, Wang H (2020) Progress in deciphering trophoblast cell differentiation during human placentation. Curr Opinion in Cell Biology 67:86-91. https://doi.org/10/j.ceb.2020.08.010. Tables Tables 1 and 2 are available in the supplementary files section Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8584838","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":579343819,"identity":"7ee3e41c-7813-4968-b324-69daf820d2fb","order_by":0,"name":"Jamal Dakour","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Jamal","middleName":"","lastName":"Dakour","suffix":""},{"id":579343820,"identity":"bd7295e2-14a9-4c53-859b-8eaa7b2e0e0b","order_by":1,"name":"Donald W. Morrish","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3PMQuCQBjG8VcEJ831XOorHAjRx7lDsEVCcGkIOQhqbLVvkQTNBy/U4hyCEEVrg9DSEFFCW3HY1nD/4ab78dwB6HT/mGwOCswFMPA34gmAhpCWBIBR2ZZ09ri9juN05JfBEePxIXWFeapVxKvCYFlQTPplSDErEkKk5SunaBX5hqCSb0pG0ZkxAtJWv+5NUr7OhjU6D0Z60jZvLYjJVyR6rQhGqLQt5UrzlxdBnhWXGLMt83K0+gMV6VQBGuKe8sV8mJ/jCXO7u+m5VJEvmT/e1+l0Ot1nT/G7TvD2H9XOAAAAAElFTkSuQmCC","orcid":"","institution":"University of Alberta","correspondingAuthor":true,"prefix":"","firstName":"Donald","middleName":"W.","lastName":"Morrish","suffix":""}],"badges":[],"createdAt":"2026-01-12 19:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8584838/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8584838/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103049570,"identity":"33d3b5a9-87ca-4d5a-ace2-80d0679b7f96","added_by":"auto","created_at":"2026-02-20 07:42:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292439,"visible":true,"origin":"","legend":"\u003cp\u003eNormal tissue distribution of PL48\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/412befbe8fe9dc125ecf5140.png"},{"id":102833304,"identity":"c49a98b5-1546-4c38-94e3-fa4591d56786","added_by":"auto","created_at":"2026-02-17 10:27:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":761507,"visible":true,"origin":"","legend":"\u003cp\u003ePL48 in situ hybridization, first trimester placenta. Dark stain shows PL48 expression\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/cb313a4e8438ac043b05d587.png"},{"id":102962990,"identity":"65209230-0fd5-40b8-8d49-9abaefe25668","added_by":"auto","created_at":"2026-02-19 04:12:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":895058,"visible":true,"origin":"","legend":"\u003cp\u003ePL48 in situ hybridization term placenta. Dark stain shows PL48 expression\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/4191c5c216cdf608bdfe5bc7.png"},{"id":102833314,"identity":"22ef0625-34f6-4998-875e-b953bf3cadd4","added_by":"auto","created_at":"2026-02-17 10:27:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":221056,"visible":true,"origin":"","legend":"\u003cp\u003ea – PL48 PCR: term (left 2 lanes), first trimester (right 2 lanes)\u003c/p\u003e\n\u003cp\u003eb – PL48 PCR term (left 2 lanes) and first trimester (right 2 lanes) placenta\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/c0f79a6e2bc6f21e061aac21.png"},{"id":102963111,"identity":"02b7f5bc-04e3-47f1-9bcc-5366324861b1","added_by":"auto","created_at":"2026-02-19 04:13:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":356961,"visible":true,"origin":"","legend":"\u003cp\u003eTUNEL labeling of MCF-7 cells. Arrows- TUNEL positive cells\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/3fbe311957579f1fb48f572b.png"},{"id":102963410,"identity":"67e67b52-4cb0-4d48-a97f-040d08d13ed9","added_by":"auto","created_at":"2026-02-19 04:17:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84640,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PL48, p53 and 21 in wild type MCF-7 and MCF-7stably transfected with PL48\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/5678398dad7e4167f3fc1503.png"},{"id":102833310,"identity":"2d407c26-9f7f-40a4-a196-aeea7bc64750","added_by":"auto","created_at":"2026-02-17 10:27:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":135832,"visible":true,"origin":"","legend":"\u003cp\u003eEffects on induction of PL48 by gamma radiation (top panel) , hypoxia, peroxynitrite, and xanthine/xanthine oxidase (middle panel) and cytokines lane 1= control, lane 2=EGF, lane 3=TGFβ, lane 4 = TNFα, lane 5 = IFNγ (bottom panel). C=control, no added substance/cytokine, T = added substance/cytokine\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/95ec60e07a2b716e4b62f865.png"},{"id":102833316,"identity":"58efef02-538c-4737-81db-09595d33fa7f","added_by":"auto","created_at":"2026-02-17 10:27:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":385487,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of antisense PL48 on syncytial formation in differentiating trophoblast\u003c/p\u003e\n\u003cp\u003eC= cytotrophoblast; S = syncytial units, outlined by desmoplakin staining\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/19be190522a790a699c62621.png"},{"id":108007124,"identity":"79bcf265-4c69-4ff3-93c9-51942e19e1da","added_by":"auto","created_at":"2026-04-28 12:58:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3527818,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/cc0a2093-0850-4756-97a4-c53003d76ba0.pdf"},{"id":102962579,"identity":"a1c250b5-b2a4-46cd-ac96-ce248d7f989b","added_by":"auto","created_at":"2026-02-19 04:09:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16816,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8584838/v1/cbc7563ad6bda6f59c3f78e1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"PL48: mechanism of a new cell growth regulatory gene that inhibits proliferation and promotes differentiation in human cytotrophoblast","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA key step in the development of the placenta is the formation of the syncytium from several precursor types of cytotrophoblast cells (Liu 2018;Lawless 2023). In human placenta, multiple factors and cell signaling pathways regulate cytotrophoblast fusion into syncytium (Lawless 2023; Io 2021; Xiao 2020; Castel \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Knofler 20219) and can be mimicked in vitro by cell reprogramming and regulation of stem cells in vitro (Castel \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Activation of apoptosis (caspase 8) is also required for cytotrophoblast differentiation (Huppertz 2001; Black 2004). In support of this concept, we found that the cytokine PL74 (MIC-1) had a bifunctional capacity to induce both differentiation and apoptosis, suggesting such factors may act as switches between differentiation and apoptosis (Li 2005).\u003c/p\u003e \u003cp\u003eIn a search for additional regulators of trophoblast differentiation, we cloned the novel gene PL48 from a subtractive library of in vitro differentiating normal human cytotrophoblast cells (Morrish et al \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Dakour et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). These cells spontaneously exit the cell cycle and differentiate into a syncytium and are a model of spontaneously differentiating trophoblast Morrish et al \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). PL48 has no similarities to known functional gene classes and initial studies demonstrated that it had low or absent expression in 6 different tumor cell types (Dakour et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). PL48 has also been found as part of genome cloning projects in man and mouse and is localized to chromosome 6 (Jea Kawai \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Because of the association of PL48 with growth arrest and differentiation, we wished to directly determine its interaction with p53 and the resultant outcome on cell proliferation, apoptosis, and differentiation in several cell models including normal human term trophoblast, MCF-7 and MDA-MB231 breast cancer cells, which have wild type p53 and mutated nonfunctional p53 respectively (Bartek 1990).\u003c/p\u003e \u003cp\u003eCells exiting the cell cycle may undergo various fates including entering a resting state, differentiation, or cell death. A key switch in this process is p53. Multiple environmental stress signals including hypoxia, DNA damage, drugs, serum deprivation, UV and gamma radiation, aberrant oncogene expression and oxidative stress can stabilize p53 protein expression leading to growth arrest and/or apoptosis (Cox \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Fridman \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Ashcroft 2000; Oren \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Oren et al 2002; Ueda 2002; Fritz 2003). It is not yet clearly understood how p53, once activated, can direct a cell to one of many fates including transient or permanent growth arrest, accelerated DNA repair, terminal differentiation, or apoptosis (Fridman \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Oren \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Oren, Damalas 2002). There are a large number of downstream target genes of p53 including the cell cycle regulator p21\u003csup\u003eCip1\u003c/sup\u003e, and both induced and repressed genes are required for apoptosis (Weber \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Furthermore, there can be both p53-dependent and independent inhibition of the cell-cycle (Kastan \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Aberrant control of these growth processes may lead to cancer and because of its pivotal position, p53 is frequently found to be mutated in cancers. In these studies, we show that PL48 has both p53-dependent and independent modes of action in regulation of cell cycle arrest and apoptosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue Distribution and Subcellular Localization\u003c/h2\u003e \u003cp\u003eThe study has received approval of the institutional ethics board. A normal human tissue dot blot representing most human tissues was obtained from Clontech Laboratories Inc., Palo Alto, CA, and probed with a full-length PL48 cDNA probe (see below). Subcellular localization of PL48 in normal first trimester and term placenta samples was performed by in situ hybridization. First trimester and term normal placental tissue were obtained from therapeutic terminations of pregnancy and normal deliveries, respectively, and snap frozen in liquid nitrogen and stored at -80\u003csup\u003eo\u003c/sup\u003e C until use. In situ hybridization was performed by first fixing small pieces of frozen placenta or breast cancer tissue in 4% paraformaldehyde in active DEPC overnight, followed by 30% sucrose overnight. 5\u0026ndash;10 \u0026micro;M sections were cut onto Super-frost slides (Fisher), air dried and stored at -80\u003csup\u003eo\u003c/sup\u003eC until use. For hybridization, slides were treated with proteinase K for 30 min at room temperature, postfixed with 4% paraformaldehyde in DEPC-PBS for 10 min, treated with triethanolamine-acetic anhydride for 10 min, washed in PBS-DEPC, and equilibrated in 5xSSC for 10 min. Slides were prehybridized for 2 h at 65\u003csup\u003eo\u003c/sup\u003eC in 50% formamide, 25% 20xSSC and salmon testes DNA (Invitrogen). The PL48 cDNA probes were constructed by PCR using the sense primer \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAA TTT AAT ACG ACT CAC TAT AGG GCG\u003c/span\u003e AGC TGG CTT TGC ACG CCT CT (T7 promoter underlined) or the antisense primer \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAA TTG GAT TTA GGT GAC ACT ATA GAA TAC\u003c/span\u003e GCT GGT GTC ATC CAT ACC CTC AT (SP6 promoter underlined). DNA products were purified on 1% agarose gels, electroeluted and purified with QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer\u0026rsquo;s instructions. Probes were denatured at 80\u003csup\u003eo\u003c/sup\u003eC for 5 min then labeled with digoxigenin using a kit (Roche) according to the manufacturer\u0026rsquo;s instructions. Probes were applied in prehybridization solution to the slides using a Mound Hybridization Sealing System (Molecular Probes) overnight at 65\u003csup\u003eo\u003c/sup\u003eC. After hybridization, slides were washed with 2xSSC for 30 min at 37\u003csup\u003eo\u003c/sup\u003eC, 2xSSC for 60 min at 65\u003csup\u003eo\u003c/sup\u003eC, then 0.1xSSC for 60 min at 65\u003csup\u003eo\u003c/sup\u003eC. Antibody solution (1:2000) for digoxigenin was added for 2 h at 37\u003csup\u003eo\u003c/sup\u003eC, the slides washed, then color developed with NBT/BCIP solution at 37\u003csup\u003eo\u003c/sup\u003eC. Slides were dehydrated in increasing 35\u0026ndash;100% ethanol solutions, counterstained with Vector Nuclear Fast Red (Vector Lab), and photographed.\u003c/p\u003e \u003cp\u003eSemi-quantitative PCR was performed to compare first trimester and term placental expression of PL48. The same protocol as above was used and primers for PL48 were as above. The annealing temperature used was 60\u003csup\u003eo\u003c/sup\u003eC. An internal standard using GAPDH was used as amplification control. Primers for GAPDH were: sense: 51-GAA GGT GAA GGT CGG AGT C-31; antisense: 51-GAA GAT GGT GAT GGG ATT TC\u0026thinsp;\u0026minus;\u0026thinsp;31. Reverse transcription was performed using the Superscript II Reverse Transcription Reagents kit (GIBCO BRL) according to the manufacturer\u0026rsquo;s instructions. The resultant cDNA mixture was stored at -20\u003csup\u003eo\u003c/sup\u003eC until ready for PCR. As a negative control, the reaction was also performed in the absence of reverse transcriptase in randomly chosen samples. The PCR reaction was performed using the Tag DNA Polymerase kit (GIBCO BRL). The PCR mixture was initially denatured at 95\u003csup\u003eo\u003c/sup\u003eC for 10 min, followed by 40 cycles of 95\u003csup\u003eo\u003c/sup\u003eC for 30 s, 60\u003csup\u003eo\u003c/sup\u003eC for 60 s, and 72\u003csup\u003eo\u003c/sup\u003eC for 30 s, with an extension cycle at 72\u003csup\u003eo\u003c/sup\u003eC for 10 min. The PCR products along with a nucleotide ladder were run on a 2% agarose gel treated with ethidium bromide and photographed under ultraviolet light.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eBecause term human cytotrophoblast cells do not proliferate in vitro (Morrish et al \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), and existing first trimester cytotrophoblast cell lines (HTR8/SVneo and SGHPL-4) have blocked p53 due to their creation using large T antigen (Aboagye-Mathiesen 1996), we chose to use breast cancer cell lines in which wild-type p53 estrogen-receptor positive MCF-7, and estrogen-receptor negative p53-mutated MDA-MB231 breast cancer cells (obtained from the American Type Culture Collection, Rockville, MD) were available and therefore could be used to determine p53-dependent and p53-independent effects. These cells were grown in 24 well multiwell plates (Corning, New York) in phenol red-free DMEM (Sigma Chemical Co., St. Louis, MO) containing 10% charcoal-stripped fetal calf serum containing 50 \u0026micro;g/ml penicillin-streptomycin (Sigma).\u003c/p\u003e \u003cp\u003eTerm and first trimester placentas were obtained from normal deliveries and therapeutic terminations of pregnancy. Tissue for in situ hybridization was snap frozen in liquid nitrogen and stored at -70\u003csup\u003eo\u003c/sup\u003e C. Term trophoblast cultures were performed as previously described using a trypsin-DNAse I digestion that produces a cell preparation over 95% pure for cytotrophoblast with fewer than 5 vimentin-positive cells per 10\u003csup\u003e5\u003c/sup\u003e cells and which is essentially free of syncytial fragments and identical in purity and function to CD9/HLA class I/II immunopurified cells (Morrish et al \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Guilbert 2002; Dakour et al \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Cells were plated at 6\u0026ndash;8 x 10\u003csup\u003e6\u003c/sup\u003e cells/dish in 100 mm Petrie dishes (Corning) for mRNA studies and cultured in 10% FBS- DMEM-penicillin-streptomycin as described (Guilbert 2002). Cells were attached for 2 h, then the medium changed to serum-free DMEM. These cells do not proliferate but rapidly differentiate spontaneously over 18\u0026ndash;24 h toward a syncytial phenotype including upregulation of most syncytial gene products and formation of morphological syncytium, but have a villous cytotrophoblast phenotype in early (\u0026thinsp;~\u0026thinsp;\u0026lt;\u0026thinsp;3 h) culture (Morrish et al \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Dakour et al \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Garcia-Lloret 1996).\u003c/p\u003e\n\u003ch3\u003eInduction of PL48 Expression\u003c/h3\u003e\n\u003cp\u003eTo determine inducers of PL48 expression, MCF-7 and normal trophoblast cells were used. Term trophoblast cultures were exposed to 18 uM peroxynitrite or 5 \u0026micro; units/ml xanthine oxidase/100 \u0026micro;M xanthine for 18 h to induce an oxidative stress, or 2% oxygen for 18 h using an hypoxia incubator (Biospherix PRO-OX model 110 regulator and chamber) to induce an hypoxic stress. DNA damage was induced by exposing MCF-7 cells for 18 h to 10 cGy gamma radiation. Cytokine effects were determined by adding 10 ng/ml EGF, 10 ng/ml TGFβ1, 10 ng/ml TNFα or 100 U/ml IFNγ (Upstate) or controls to culture medium of term cytotrophoblast cultures for 24 h. Four separate experiments were performed for each of hypoxia, peroxynitrite and xanthine/xanthine oxidase, and two experiments for cytokine effects.\u003c/p\u003e\n\u003ch3\u003eTransfections\u003c/h3\u003e\n\u003cp\u003eA full length insert of PL48 as previously reported (Dakour et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) was cloned into pcDNA3 for transient transfections of MCF-7 and MDA-MB231 cells. Transient transfections were performed using Lipofectin by adding 0.5 \u0026micro;g/ml pcDNA3-PL48 or 0.5 \u0026micro;g/ml pcDNA3 alone (control) DNA to cells. Three days after transfection, cells were evaluated comparing control untransfected cells, empty vector alone and vector-PL48 as follows: (1) cell counts using a hemocytometer, (2) \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake, and (3) apoptosis quantitation using the TUNEL method. Three separate transient transfections of MCF-7 and two of MDA-MB231 cells were evaluated. Transfection efficiency was determined in MCF-7 and HL-60 cells (see below) by co-transfecting the β-galactosidase gene in pCMV, staining transfected cells with X-Gal, and counting blue-stained (positively-transfected) cells (In Situ β-Galactosidase Staining Kit, Stratagene Cloning Systems, La Jolla, CA).\u003c/p\u003e \u003cp\u003eStable transfectants of PL48 with an ecdysone-inducible promoter were prepared according to the manufacturer\u0026rsquo;s instructions (Ecdysone-Inducible Expression Kit, In Vitrogen, San Diego, CA). In brief, PL48 was cloned into the multiple cloning site of pIND and the resulting construct co-transfected with the pVgRXR vector into MCF-7 cells. Transfected cells were selected by using double selection with ampicillin and Zeocin as indicated by the manufacturer. The PL48-transfected cells were assessed for induction of PL48 by adding 1 \u0026micro;M of the ecdysone analogue muristerone A to the medium for 24 hours and then harvesting the cells for mRNA and northern blot analysis as described below. \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake was performed on uninduced cells and muristerone A-induced cells. We also studied induced and uninduced cells to which 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M tamoxifen or 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e M estradiol had been added to determine if estrogen or estrogen-receptor mediated events could induce PL48 in MCF-7 cells. Stable transfectants were also constructed in MCF-7 and MDA-MB231 cells using a retroviral vector (Clontech, Tet Off), according to the manufacturer\u0026rsquo;s instructions. Induction of the promoter was achieved by removing tetracycline from the culture medium.\u003c/p\u003e\n\u003ch3\u003eEffects of PL48 on Human Cytotrophoblast Differentiation\u003c/h3\u003e\n\u003cp\u003eTo determine the effects of PL48 on human cytotrophoblast differentiation, a full-length antisense construct of PL48 was prepared in pcDNA3. The antisense construct or empty vector control was transiently transfected as above into term primary human cytotrophoblast cultures (prepared by CD9/HLA class I/II immunopurification) immediately after attachment of cells. Transfection was allowed to proceed for 72 h at which time the cultures were stopped and fixed for immunostaining for desmoplakin. Desmoplakin staining was performed as previously described (Douglas \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Formation of syncytia was quantitated by counting nuclei within desmoplakin-stained outlines (Douglas \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The ratio of multinucleated cell groups (at least two nuclei) to total number of nuclei was calculated as a measure of syncytial unit formation and expressed as a percentage. Two separate transfection experiments were performed immediately after thawing and reconstituting two different cell preparations. Seven randomly chosen microscope fields were counted in total to determine percent syncytial unit formation, and 78\u0026ndash;125 nuclei per field were counted in each experiment.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003e3\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eH-thymidine uptake\u003c/em\u003e \u003c/p\u003e \u003cp\u003e1 \u0026micro;Ci \u003csup\u003e3\u003c/sup\u003eH-thymidine was added for 3 h to quadruplicate wells of MCF-7 or MDA-MB231 cells. The cells were then washed three times with PBS, briefly trypsinized with 0.25% trypsin, and cells centrifuged for 3 min at 300 x g. The cell pellet was dissolved in liquid scintillation cocktail (EcoLite, ICN Biochemicals, Costa Mesa, CA) for liquid scintillation counting.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL assay of DNA nicking (apoptosis)\u003c/h2\u003e \u003cp\u003eThis assay detects nuclear DNA fragmentation in apoptotic cells using terminal deoxynucleotidyl transferase (TdT)-mediated DTU)-biotin DNA-nick end labeling of free 3'-OH termini and was performed using a kit (In Situ Death Detection Kit, AP, Boehringer Mannheim Canada, Laval, PQ) following the manufacturer\u0026rsquo;s instructions. Three separate microscope fields containing at least 150 cells of each of control or PL48-transfected MCF-7 or MDA-MB231 cells were counted for TUNEL-positive cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNorthern blot analysis\u003c/h3\u003e\n\u003cp\u003emRNA was extracted by use of RNeasy Mini Kit (QIAGEN Inc., Mississauga, ON). Fifteen micrograms of the extract were separated on a 1.2% agar-formaldehyde denaturing gel and transferred to Nytran Plus membrane (Schleicher \u0026amp; Schuell, Keene, NH) by standard methods (Sambrook 1989). The blot was probed with a full length PL48 cDNA probe labeled by random priming labeling using a DNA Labeling System (GIBCO BRL Life Technologies, Gaithersburg, MD) as previously described (Morrish, Linetsky 1996). RNA loading was quantitated by reprobing the blots with 18 S rRNA and performing densitometry.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eTissue distribution and Localization of PL48\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll normal human tissues tested expressed PL48mRNA (Fig 1). \u0026nbsp;In situ hybridization of PL48 in placenta showed expression exclusively in syncytiotrophoblast in a nuclear and perinuclear distribution \u0026nbsp;(Figs 2,3). \u0026nbsp;Expression was quite weak in the first trimester (Fig 2) but strong at term (Fig 3), indicating gestational dependence of expression. \u0026nbsp;PL48 expression by semi-quantitative PCR confirms this gestational difference (Figs 4a,4b).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunction and signaling pathway of PL48\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe transiently transfected PL48 into MCF-7 and MDA-MB231 cells and measured \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake, cell number, and apoptosis using TUNEL (Table 1). \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake decreased significantly (\u003csup\u003e3\u003c/sup\u003eH-thymidine uptake: MCF-7: p\u0026lt;0.05; MDA-MB231: p\u0026lt;0.05). \u0026nbsp;Cell number also decreased significantly. \u0026nbsp; \u0026nbsp;Quantitation of apoptotic cells using TUNEL showed a significant increase after PL48 transfection into MCF-7 cells (control: 5.1\u003cu\u003e+\u003c/u\u003e0.2%; PL48: 16.0\u003cu\u003e+\u003c/u\u003e1.4%; t-test, p \u0026lt; 0.025; Fig 5) but no induction of apoptosis after transfection into MDA-MB231 cells (control 5.5\u003cu\u003e+\u003c/u\u003e1.0%, PL48 5.0\u003cu\u003e+\u003c/u\u003e1.1% p = NS; Fig 5). \u0026nbsp; Transfection efficiency as determined by co-transfection of galactosidase was 15-20%. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInitial attempts to stably transfect PL48 using pcDNA3 were unsuccessful because PL48 expression was lethal. \u0026nbsp;We therefore made stable transfectants in MCF-7 cells using an ecdysone-inducible promoter system. \u0026nbsp;In this preparation, PL48 expression was significantly induced using the ecdysone analogue muristerone A. \u0026nbsp;Wild-type cells expressed very low or absent levels of PL48 not detectable on northern blot (Fig 6). Reprobing the same blot with p53 and p21 demonstrated that both genes \u0026nbsp;were strongly induced in MCF-7 transfected cells compared to untransfected cells. \u0026nbsp; \u0026nbsp;We also stably transfected MCF-7 and MDA-MB231 cells with a retroviral vector with an inducible promoter (Clontech, Tet Off). \u0026nbsp;After promoter induction, PL48 induced a 37-63% decrease in \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake in these cells (Table 2). \u0026nbsp;Presumably because of incomplete suppression of promoter activity, transfected cells with uninduced promoter showed 17-44% reduction in \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake.\u003c/p\u003e\n\u003cp\u003eTo determine if PL48 acted through the estrogen receptor, ecdysone-inducible stably transfected MCF-7 cells were exposed to 10\u003csup\u003e-9\u003c/sup\u003e M estradiol or 10\u003csup\u003e-6\u003c/sup\u003e M tamoxifen, both with and without muristerone A induction and compared to control untransfected cells (Table 2). \u0026nbsp; Induced cells had approximately 20% reduction in\u003csup\u003e\u0026nbsp;3\u003c/sup\u003eH-thymidine uptake, similar in magnitude to the effect of tamoxifen during this exposure period. \u0026nbsp;Estradiol induced a large increase in \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake, but concomitant PL48 expression still resulted in about a 20% reduction of the growth-stimulatory effect of estradiol.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInduction\u003c/em\u003e \u003cem\u003eof PL48 Expression\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine if DNA damage were an inducer of PL48, MCF-7 cells were exposed to 10 cGy of gamma irradiation for up to 18 h (Fig 7, top panel). \u0026nbsp; No induction was observed. \u0026nbsp;We also tested the effects of 2% hypoxia and oxidative stress (induced by peroxynitrite and xanthine/xanthine oxidase) in term trophoblast \u0026nbsp;(Fig 7, middle panel). \u0026nbsp;Severe oxidative stress as induced by peroxynitrite, but not weaker oxidative stress induced by xanthine/xanthine oxidase nor hypoxia, induced PL48. \u0026nbsp; Previously, we have shown that the non-physiologic agent DMSO will also induce PL48 in HL-60 cells, but this is presumed to be a concomitant of differentiation and not a direct inducer of PL48 (Morrish et al 1991). Exposure of trophoblast cells to EGF, TNFα, TGFβ, or INFγ had no effect on PL48 expression (Fig 7, bottom panel). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEffects of PL48 on Cytotrophoblast Differentiation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTransfection of antisense PL48 reduced spontaneous cytotrophoblast differentiation into syncytial units by 19.9+1.1% (p\u0026lt;0.05, ANOVA) (Fig 8).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe first determined the expression and localization of PL48 in human tissues, and found that PL48 was widely distributed in all normal tissues tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In situ hybridization and semi-quantitative PCR demonstrated that placental expression was restricted to syncytium, with a gestational dependence in which term syncytium showed higher expression compared to first trimester (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBecause primary term human cytotrophoblast cells do not proliferate, and first trimester cytotrophoblast cell lines (eg HTR8/SVneo) have mutated p53, we elected to study effects of PL48 on cell proliferation and apoptosis using two breast cancer cell lines, MCF-7 and MDA-MB231, as model systems of wild-type and mutated p53 respectively, We found that PL48 overexpression by transfection significantly inhibited cell proliferation regardless of p53 status, up to 63% when using a retroviral vector. During chronic exposure in stably transfected cell lines, we also found that PL48 displayed a lethal phenotype when highly expressed, requiring the use of suppressible promoter systems to allow generation of stable transfectants. The inhibition of growth in MCF-7 cells was not through the estrogen receptor because blocking of the receptor with tamoxifen did not prevent proliferation and the effect occurred in MDA-MB231 cells which lack the estrogen receptor.\u003c/p\u003e \u003cp\u003eTo investigate further the mechanism of reduction in cell growth in MCF-7 cells, we assessed the effects of PL48 on apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These data demonstrated that PL48 significantly induced apoptosis in MCF-7 cells but not in MDA-MB231 cells. Since MCF-7 cells contain a wild-type p53 but MDA-MB231 cells have a nonfunctional mutated p53 (Fridman \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), the data indicate PL48 requires functional p53 to induce apoptosis. Stable overexpression of PL48 in MCF-7 cells resulted in induction of p53 and p21 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003e). P53 transcriptionally activates p21, thus leading to cell cycle arrest (Cox \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Thus, PL48 can act through this pathway to inhibit cell proliferation. However, it is known that p21 expression can also be induced independently of p53 by a variety of factors including MyoD, NGF, oxygen radicals and TGFβ (Cox \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Since PL48 also caused growth inhibition in MDA-MB231 cells which lack a functional p53 and are unable to induce p21, the data would indicate PL48 is also able to bypass p53 by an alternate pathway (Oren \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Huo \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Chen 1996) to directly induce p21 and cell cycle arrest. P53 can mediate apoptosis signals (Waldman 1996) and this can occur in cells lacking p21 (Polyak 1996; Agarwal 1998), but the precise pathway or mechanism of this effect is unknown (Cox \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Chen 1996). In the model systems we used, however, PL48 appears to require p53 to cause apoptosis, since MDA-MB231 cells do not undergo this process when PL48 is overexpressed.\u003c/p\u003e \u003cp\u003eSeveral genotoxic stresses are known to induce p53 resulting in apoptosis (Ashcroft 2000; Ueda 2002; Fritz 2003; Weber \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Agarwal 1998). To determine if any of these factors induced PL48, we tested gamma irradiation on MCF-7 cells, and 2% hypoxia, cytokines (TNFα, TGFβ, and IFNγ) and oxidative stress on trophoblast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Of these, only strong oxidative stress (peroxynitrite) induced PL48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e). TNFα and IFNγ are known to induce apoptosis in trophoblast cells (Douglas \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) but such actions would not appear to involve PL48 induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince PL48 expression is coincident with cytotrophoblast differentiation into syncytium, we tested whether PL48 expression could mediate this differentiation process. We found that antisense PL48 transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003e) significantly inhibited spontaneous cytotrophoblast differentiation as assessed by the formation of desmoplakin-defined syncytial units. Since PL48 has high expression in placental syncytium, the results imply that PL48 may contribute to the induction and maintenance of the differentiated syncytial state. We have previously shown that EGF induces (Morrish et al \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) and TGFβ1 inhibits (Morrish et al \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) cytotrophoblast differentiation. However, neither of these cytokines altered PL48 expression, indicating their action in modulating differentiation was not through PL48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The actions of PL48 to regulate not only differentiation but also apoptosis and proliferation appear analogous to similar properties of the cytokine PL74 (MIC-1) as we have demonstrated (Li et al \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Huppertz et al have shown that human cytotrophoblast differentiation into syncytium are coupled, with inactive apoptotic proteins being present in cytotrophoblast, and these proteins then become activated with an obligatory activation of caspase 8 when the cells fuse to form syncytium (Huppertz 2001; Black 2004). In this context, cytokines and genes such as PL74 (MIC-1) and PL48 thus can regulate both apoptosis and differentiation and so may control the degree to which each process occurs (Li et al \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePL48 thus appears to be a new promoter of cytotrophoblast differentiation that also has capabilities to induce apoptosis or cell cycle arrest depending on whether wild-type or mutated p53 is present in a cell. It is also an upstream regulator of p53, possibly acting in response to severe cellular oxidative stresses. PL48 effects are particularly potent on growth inhibition. Whether the end result of PL48 action is apoptosis or only growth arrest appears to depend at least in part on the functional status of p53. Since the ultimate result of p53 activation (growth arrest, apoptosis, terminal differentiation) is thought to be dose-dependent on the degree of p53 activation (Oren \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) this may therefore be a factor in determining which PL48 effects take place.\u003c/p\u003e \u003cp\u003eAs PL48 is expressed in many human tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e) the data imply PL48 may have similar functions of growth inhibition, apoptosis and growth regulation in diverse tissues.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by the Canadian Institutes of Health Research. We wish to thank Larry J Guilbert, PhD, Departments of Medicine and Medical Microbiology and Immunology for the CD9/HLA I/II purified cells and Hongshi Li, MSc, (current address Kinda Bioengineering, Yueyang, PR, China), Bonnie Winkler-Lowen BSc, Dept. of Medical Microbiology and Immunology, \u0026nbsp;and Xin Fang, PhD, Perinatal Research Centre, University of Alberta,, for technical assistance.\u003c/p\u003e\n\u003cp\u003eContributors: I declare that I participated in the design and conduct of the study. \u0026nbsp;J Dakour conducted most of the studies. DW Morrish designed the study and conducted some of the work. \u0026nbsp;There is no conflict of interest by either author.\u003c/p\u003e\n\u003cp\u003eDeclaration of interests : \u0026nbsp;none\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAboagye-Mathiesen G, Zdravkovic M, Toth ED, Graham CH, Lala PK, Ebbesen P (1996) Altered expression of the tumor suppressor/oncoprotein p53 in SV40 Tag-transformed human placental trophoblast and malignant trophoblast cell lines. Early Pregnancy 22:102-12.\u003c/li\u003e\n \u003cli\u003eAgarwal ML, Taylor MR, Chernov MV, Chernova OB, Stark GR (1998) The p53 network J Biol Chem, 271:1-4.\u003c/li\u003e\n \u003cli\u003eAshcroft M, Taya Y, Vousden KH (2000) Stress signals utilize multiple pathways to stabilize p53. Mol Cell Biol 20:3224-33.\u003c/li\u003e\n \u003cli\u003eBartek J, Iggo R, Gannon J, Lane DP (1990) Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines. 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J Clin Endocrinol Metab 90:3045-3053.\u003c/li\u003e\n \u003cli\u003eLiu Y, Fan X, Wang R, Lu X, Dang Y-L, Wang H, Lin H-Y, Zhu C, Ge H, Cross JC, Wang H (2018) Single-cell RNA-seq reveals the diversity of trophoblast subtypes and patterns of differentiation in the human placenta. Cell Research, 28:819-832. https://doi.org/10.1038/s41422-018-0066-y\u003c/li\u003e\n \u003cli\u003eMorrish DW, Bhardwaj D, Dabbagh LK, Marusyk H, Siy O (1987) Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J Clin Endocrinol Metab 65:1282-90.\u003c/li\u003e\n \u003cli\u003eMorrish DW, Bhardwaj D, Paras MT (1991) Transforming growth factor b1 inhibits placental differentiation and human chorionic gonadotropin and human placental lactogen secretion. Endocrinology 129:22-26.\u003c/li\u003e\n \u003cli\u003eMorrish DW, Dakour J, Li H, Xiao J, Miller R, Sherburne R, Berdan RC, Guilbert LJ (1997) In vitro cultured human term cytotrophoblast: \u0026nbsp;a model for normal primary epithelial cells demonstrating a spontaneous differentiation program that requires EGF for extensive development of syncytium. Placenta 18:577-85.\u003c/li\u003e\n \u003cli\u003eMorrish DW, Linetsky E, Bhardwaj D, Li H, Dakour J, Marsh RG, Paterson MC, \u0026nbsp;Godbout R (1996) Identification by subtractive hybridization of a spectrum of novel and unexpected genes \u0026nbsp;associated with in vitro differentiation of human cytotrophoblast cells. Placenta 17:431-41.\u003c/li\u003e\n \u003cli\u003eOren M (2003) Decision making by p53: \u0026nbsp;life, death and cancer. Cell Death Differ 10:431-42.\u003c/li\u003e\n \u003cli\u003eOren M, Damalas A, Gottlieb T, Michael D, Taplick J, Leal JFM, Maya R, Moas M, \u0026nbsp;Seger R, Taya Y, Ben-Ze\u0026rsquo;ev A (2002) Regulation of p53. \u0026nbsp;Intricate loops and delicate balances. Annals NY Acad Sci 973:374-383\u003c/li\u003e\n \u003cli\u003ePolyak K, Waldman T, He TC, Kinzler KW, Vogelstein B (1996) Genetic determinants of p53-induced apoptosis and growth arrest. Genes Dev 10:1945-52.\u003c/li\u003e\n \u003cli\u003eSambrook J, Fritsch EF , Maniatis T (1989) Extraction, purification, and analysis of messenger RNA from eukaryotic cells. Sambrook J, Fritsch EF, Maniatis T. Cold Spring Harbor Press\u003c/li\u003e\n \u003cli\u003eUeda S, Masutani H, Nakamura H, Tanaka T, Ueno M, Yodoi J (2002) Redox control of cell death. Antioxid Redox Signal 4:405-14\u003c/li\u003e\n \u003cli\u003eWaldman T, Lengauer C, Kinzler KW, Vogelstein B (1996) Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381:713-16.\u003c/li\u003e\n \u003cli\u003eWeber JD, Zambetti GP (2003) Renewing the debate over the p53 apoptotic response. Cell Death Differ 10:409-12.\u003c/li\u003e\n \u003cli\u003eXiao Z, Yan L, Liang X, Wang H (2020) Progress in deciphering trophoblast cell differentiation during human placentation. Curr Opinion in Cell Biology 67:86-91. \u0026nbsp;https://doi.org/10/j.ceb.2020.08.010.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the supplementary files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PL48, p53, apoptosis, placenta, differentiation","lastPublishedDoi":"10.21203/rs.3.rs-8584838/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8584838/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction:\u0026nbsp; PL48 is a novel human placental differentiation-associated gene that is widely expressed in normal tissues.\u0026nbsp; Methods:\u0026nbsp; To determine the effect and mechanism of PL48 on cell proliferation, apoptosis, and cytotrophoblast differentiation, several cell models were used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults:\u0026nbsp; Antisense PL48 transfection into term human trophoblast reduced spontaneous cytotrophoblast differentiation into syncytium by 19.9\u003cu\u003e+\u003c/u\u003e1.1 %. Transiently transfected MCF-7 cells demonstrated a 19.5-25.7% decrease in \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake concomitantly with a 20% decrease in cell number and 3-fold increase in apoptotic cells shown by TUNEL labeling.\u0026nbsp; Stable retroviral transfection of PL48 into MCF-7 cells resulted in a 50% decrease in \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake after gene promoter induction. Estrogen-induced proliferation of MCF-7 cells was not affected by PL48 induction with or without tamoxifen, indicating PL48 does not act through the estrogen receptor. Transfected MDA-MB231 cells showed a 20% decrease in \u003csup\u003e3\u003c/sup\u003eH-thymidine uptake but no induction of apoptosis indicating that a functional p53 is required for PL48-induced apoptosis. Stably transfected MCF-7 cells showed an upregulation of p53 and p21 mRNA.\u0026nbsp; Peroxynitrite, but none of xanthine/xanthine oxidase, hypoxia, or cytokines (EGF, IFNγ, TNFα, TGFβ) induced PL48 in trophoblast cells.10cGy gamma irradiation did not induce PL48 in MCF-7 cells.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDiscussion:\u0026nbsp; We conclude that PL48 has multiple actions including inhibition of proliferation independently of p53,\u0026nbsp; induction of apoptosis through the p53 pathway, and enhancing differentiation in cytotrophoblast.\u003c/p\u003e","manuscriptTitle":"PL48: mechanism of a new cell growth regulatory gene that inhibits proliferation and promotes differentiation in human cytotrophoblast","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 10:27:33","doi":"10.21203/rs.3.rs-8584838/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"19cae875-0670-4302-bb4f-6d3ca6b166c7","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T11:56:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 10:27:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8584838","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8584838","identity":"rs-8584838","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}