A Repertoire of Cell Cycle Regulators Whose Expression Is Coordinated with Human Cytotrophoblast Differentiation

Although placental development depends on careful coordination of trophoblast proliferation and differentiation, little is known about the mitotic regulators that are key to synchronizing these events. We immunolocalized a broad range of these regulators in tissue sections of the maternal-fetal interface (first trimester through term) that contained floating villi (which include cytotrophoblasts differentiating into syncytiotrophoblasts) and anchoring villi (which include cytotrophoblasts differentiating into invasive cells). Trophoblast populations at the maternal-fetal interface stained for 16 of the cell cycle regulators whose expression we studied. The staining patterns changed as a function of both differentiation and gestational age. Differentiation along the invasive pathway was associated with entrance into, then permanent withdrawal from, the cell cycle, as evidenced by the orchestrated expression of cyclins, their catalytic subunits, and inhibitors. Surprisingly, we found coexpression of molecules that regulate different portions of the cell cycle in the syncytium. These data, which constitute one of the few examples to date of in situ localization of an extensive repertoire of mitotic regulators, provide the basis for studies aimed at understanding factors that lead to abnormal placentation. Although placental development depends on careful coordination of trophoblast proliferation and differentiation, little is known about the mitotic regulators that are key to synchronizing these events. We immunolocalized a broad range of these regulators in tissue sections of the maternal-fetal interface (first trimester through term) that contained floating villi (which include cytotrophoblasts differentiating into syncytiotrophoblasts) and anchoring villi (which include cytotrophoblasts differentiating into invasive cells). Trophoblast populations at the maternal-fetal interface stained for 16 of the cell cycle regulators whose expression we studied. The staining patterns changed as a function of both differentiation and gestational age. Differentiation along the invasive pathway was associated with entrance into, then permanent withdrawal from, the cell cycle, as evidenced by the orchestrated expression of cyclins, their catalytic subunits, and inhibitors. Surprisingly, we found coexpression of molecules that regulate different portions of the cell cycle in the syncytium. These data, which constitute one of the few examples to date of in situ localization of an extensive repertoire of mitotic regulators, provide the basis for studies aimed at understanding factors that lead to abnormal placentation. The placenta, which forms the interface between the embryo/fetus and the mother, is a critical determinant of pregnancy outcome. A great deal of information about its unique architecture and functions has come from studies that use biopsy specimens of the maternal-fetal interface.1Damsky CH Fisher SJ Trophoblast pseudo-vasculogenesis: faking it with endothelial adhesion receptors.Curr Opin Cell Biol. 1998; 10: 660-666Crossref PubMed Scopus (216) Google Scholar The placenta, the fetal portion of this interface, is the first organ to function during development. In parallel, the uterine lining or placental bed, the maternal portion of this interface, undergoes extensive remodeling.1Damsky CH Fisher SJ Trophoblast pseudo-vasculogenesis: faking it with endothelial adhesion receptors.Curr Opin Cell Biol. 1998; 10: 660-666Crossref PubMed Scopus (216) Google Scholar, 2Cross JC Werb Z Fisher SJ Implantation and the placenta: key pieces of the development puzzle.Science. 1994; 266: 1508-1518Crossref PubMed Scopus (1171) Google Scholar, 3Rinkenberger JL Cross JC Werb Z Molecular genetics of implantation in the mouse.Dev Genet. 1997; 21: 6-20Crossref PubMed Scopus (117) Google Scholar, 4Aplin JD Haigh T Lacey H Chen CP Jones CJ Tissue interactions in the control of trophoblast invasion.J Reprod Fertil Suppl. 2000; 55: 57-64PubMed Google Scholar The human placenta’s unique anatomy is due in large part to differentiation of its epithelial stem cells, termed cytotrophoblasts (CTBs).5Fisher SJ Damsky CH Human cytotrophoblast invasion.Semin Cell Biol. 1993; 4: 183-188Crossref PubMed Scopus (189) Google Scholar How these cells differentiate determines whether chorionic villi, the placenta’s functional units, float in maternal blood or anchor the conceptus to the uterine wall. In floating villi, CTB stem cells (referred to here as villus CTBs or vCTBs) differentiate by fusing to form multinucleated syncytiotrophoblasts (STBs) whose primary function, transport, is ideally suited to their location at the villus surface. In anchoring villi, CTB stem cells also fuse, but many remain as single cells that detach from their basement membrane and form aggregates termed cell columns. CTBs at the distal ends of these columns attach to and then deeply invade the uterus (interstitial invasion) and its vessels (endovascular invasion). Interestingly, endovascular invasion is more extensive on the arterial than the venous side. During this process CTBs replace the endothelial and muscular lining of uterine vessels, a process that greatly enlarges the diameter of arterioles, initiates maternal blood flow to the placenta, and permits venous return to the maternal circulation. As in most organs, the placenta retains a pool of undifferentiated stem cells that are evident even at term. Whether they can compensate for placental damage by differentiating later in gestation is an interesting possibility that has been hard to prove. Some of the molecular mechanisms that govern human CTB differentiation and invasion are well understood. These include an upstream suite of transcriptional regulators such as Mash-2,6Guillemot F Nagy A Auerbach A Rossant J Joyner AL Essential role of Mash-2 in extraembryonic development.Nature. 1994; 371: 333-336Crossref PubMed Scopus (519) Google Scholar Hand1,7Firulli AB McFadden DG Lin Q Srivastava D Olson EN Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1.Nat Genet. 1998; 18: 266-270Crossref PubMed Scopus (293) Google Scholar, 8Riley P Anson-Cartwright L Cross JC The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis.Nat Genet. 1998; 18: 271-275Crossref PubMed Scopus (420) Google Scholar and Gcm1,9Janatpour MJ Utset MF Cross JC Rossant J Dong J Israel MA Fisher SJ A repertoire of differentially expressed transcription factors that offers insight into mechanisms of human cytotrophoblast differentiation.Dev Genet. 1999; 25: 146-157Crossref PubMed Scopus (103) Google Scholar, 10Anson-Cartwright L Dawson K Holmyard D Fisher SJ Lazzarini RA Cross JC The glial cells missing-1 gene is essential for branching morphogenesis in the chorioallantoic placenta.Nat Genet. 2000; 25: 311-314Crossref PubMed Scopus (333) Google Scholar and a downstream set of effectors such as adhesion molecules,11Damsky CH Librach C Lim KH Fitzgerald ML McMaster MT Janatpour M Zhou Y Logan SK Fisher SJ Integrin switching regulates normal trophoblast invasion.Development. 1994; 120: 3657-3666PubMed Google Scholar proteinases,12Librach CL Werb Z Fitzgerald ML Chiu K Corwin NM Esteves RA Grobelny D Galardy R Damsky CH Fisher SJ 92-kD type IV collagenase mediates invasion of human cytotrophoblasts.J Cell Biol. 1991; 113: 437-449Crossref PubMed Scopus (638) Google Scholar and the trophoblast major histocompatibility antigen HLA-G.13Kovats S Main EK Librach C Stubblebine M Fisher SJ DeMars R A class I antigen, HLA-G, expressed in human trophoblasts.Science. 1990; 248: 220-223Crossref PubMed Scopus (1204) Google Scholar, 14McMaster MT Librach CL Zhou Y Lim KH Janatpour MJ DeMars R Kovats S Damsky C Fisher SJ Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.J Immunol. 1995; 154: 3771-3778PubMed Google Scholar In comparison, much less is known about how CTB proliferation is coordinated with differentiation. Although reagents, including many antibodies, are available for studying the expression of cell cycle regulators in tissue sections, few published studies have broadly used this approach to localize markers that are specifically expressed during key transitions and phases. This information, in conjunction with the extensive mechanistic insights that have been obtained about the biochemical roles of cell cycle regulators,15Kohn KW Molecular interaction map of the mammalian cell cycle control and DNA repair systems.Mol Biol Cell. 1999; 10: 2703-2734Crossref PubMed Scopus (409) Google Scholar could be very informative. Although a few markers have been localized,16Chilosi M Piazzola E Lestani M Benedetti A Guasparri I Granchelli G Aldovini D Leonardi E Pizzolo G Doglioni C Menestrina F Mariuzzi GM Differential expression of p57kip2, a maternally imprinted cdk inhibitor, in normal human placenta and gestational trophoblastic disease.Lab Invest. 1998; 78: 269-276PubMed Google Scholar, 17Ichikawa N Zhai YL Shiozawa T Toki T Noguchi H Nikaido T Fujii S Immunohistochemical analysis of cell cycle regulatory gene products in normal trophoblast and placental site trophoblastic tumor.Int J Gynecol Pathol. 1998; 17: 235-240Crossref PubMed Scopus (38) Google Scholar, 18Bamberger A Sudahl S Bamberger CM Schulte HM Löning T Expression patterns of the cell-cycle inhibitor p27 and the cell-cycle promoter cyclin E in the human placenta throughout gestation: implications for the control of proliferation.Placenta. 1999; 20: 401-406Abstract Full Text PDF PubMed Scopus (42) Google Scholar it is not surprising that CTB progression through and exit from the cell cycle as a function of differentiation have not been systematically studied. Based on mitotic index,19Tedde G Tedde Piras A Mitotic index of the Langhans' cells in the normal human placenta from the early stages of pregnancy to the term.Acta Anatom. 1978; 100: 114-119Crossref PubMed Scopus (11) Google Scholar it is now well established that vCTBs are placental stem cells.20Benirschke K Kaufmann P Pathology of the Human Placenta. 3rd ed. Springer-Verlag, New York1995Crossref Google Scholar, 21Fox H 2nd ed. Pathology of the Placenta. vol. 7. W. B. Saunders, London1997Google Scholar In addition to vCTBs, proliferative cells that express S phase markers are also detected in the proximal portions of cell columns associated with anchoring villi.22Bulmer JN Morrison L Johnson PM Expression of the proliferation markers Ki67 and transferrin receptor by human trophoblast populations.J Reprod Immunol. 1988; 14: 291-302Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 23Arnholdt H Meisel F Fandrey K Löhrs U Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies.Virchows Arch B Cell Pathol. 1991; 60: 365-372Crossref Scopus (138) Google Scholar, 24Mühlhauser J Crescimanno C Kaufmann P Höfler H Zaccheo D Castellucci M Differentiation and proliferation patterns in human trophoblast revealed by c-erbB-2 oncogene product and EGF-R.J Histochem Cytochem. 1993; 41: 165-173Crossref PubMed Scopus (134) Google Scholar Immunostaining of first trimester placental bed biopsy specimens with an antibody against the Ki67 antigen, which is expressed by cells that are synthesizing DNA, revealed that its expression abruptly stops at sites where CTBs differentiate and attach to the uterine wall. Together, these data suggest that differentiation of invasive CTBs is coordinated with exit from the cell cycle.25Genbacev O Zhou Y Ludlow JW Fisher SJ Regulation of human placental development by oxygen tension.Science. 1997; 277: 1669-1672Crossref PubMed Scopus (740) Google Scholar The expression patterns of G1 and G2 cyclins and of their cyclin-dependant kinases (CDKs) further support this hypothesis.17Ichikawa N Zhai YL Shiozawa T Toki T Noguchi H Nikaido T Fujii S Immunohistochemical analysis of cell cycle regulatory gene products in normal trophoblast and placental site trophoblastic tumor.Int J Gynecol Pathol. 1998; 17: 235-240Crossref PubMed Scopus (38) Google Scholar To test this hypothesis, we immunolocalized markers that are specifically expressed during all of the key transitions and phases of the cell cycle in tissue sections of the maternal-fetal interface. The results showed that as CTBs differentiate/invade, they down-regulate the expression of molecules that are associated with mitosis and up-regulate the expression of a number of inhibitors that engineer permanent withdrawal from the cell cycle. In contrast, multinucleate STBs coexpressed an unusual repertoire of markers that are usually segregated to distinct portions of the cell cycle. We also saw interesting gestation-related changes. By the second trimester, many fewer CTB stem cells and cells in columns expressed mitotic markers. There was a reciprocal increase in the number of column CTBs that expressed inhibitors. Together, these data suggest that CTB proliferation, like differentiation, is part of a developmental program that is timed to precede development of the embryo/fetus. Chorionic villi with attached decidua were collected immediately after elective pregnancy terminations for nonmedical reasons or after spontaneous deliveries. Because of their relatively small size, every sample from each first trimester placenta was processed. Second and third trimester samples were from three to five randomly chosen sites. None of the placentas had abnormalities that could be detected either grossly or histologically. Tissues were obtained from women whose pregnancies were terminated at 8 to 10 weeks (3 samples), 15 to 17 weeks (7 samples), or 20 weeks of gestation (5 samples). Fifteen samples were obtained at the time of normal term delivery (38–40 weeks of gestation). Conclusions were based on analysis of all samples in each group. Antibodies were obtained from the following sources. Mouse monoclonal anti-cyclin D1, -cyclin D2, -cyclin D3, -p21, -p16, and -Mdm2, and rabbit polyclonal anti-cyclin E (C-19), -CDK4, -CDK6, -Cdc2, -p57, and -p27 were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-phospho-specific p53 was purchased from New England Biolabs (Beverly, MA). Mouse monoclonal anti-p53 and -cyclin A were from Oncogene Research Products (Cambridge, MA). Mouse monoclonal anti-cyclin D2, -cyclin B, and -pRb were purchased from Pharmingen (San Diego, CA). Rabbit polyclonal anti-histone 3 was from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal anti-Ki67 was purchased from DAKO (Carpinteria, CA). The rat anti-human cytokeratin (CK) monoclonal antibody (7D3) was produced by the Fisher laboratory.11Damsky CH Librach C Lim KH Fitzgerald ML McMaster MT Janatpour M Zhou Y Logan SK Fisher SJ Integrin switching regulates normal trophoblast invasion.Development. 1994; 120: 3657-3666PubMed Google Scholar Rhodamine- and fluorescein-labeled goat anti-mouse, -rat, and -rabbit IgG were obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). The specificities of antibodies that had not been previously used for immunolocalization were studied further. These included anti-cyclin D1, -cyclin B, -p21, -Mdm2, -p57, -p27, and -pRb, whose reactivity with a band of the expected molecular weight was confirmed by immunoblotting samples of electrophoretically separated human chorionic villi and purified CTB lysates as previously described.25Genbacev O Zhou Y Ludlow JW Fisher SJ Regulation of human placental development by oxygen tension.Science. 1997; 277: 1669-1672Crossref PubMed Scopus (740) Google Scholar Placental tissues were processed for double indirect immunolocalization as previously described.26Damsky CH Fitzgerald ML Fisher SJ Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo.J Clin Invest. 1992; 89: 210-222Crossref PubMed Scopus (604) Google Scholar, 27Zhou Y Damsky CH Chiu K Roberts JM Fisher SJ Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts.J Clin Invest. 1993; 91: 950-960Crossref PubMed Scopus (504) Google Scholar, 28Genbacev O Joslin R Damsky CH Polliotti BM Fisher SJ Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in preeclampsia.J Clin Invest. 1996; 97: 540-550Crossref PubMed Scopus (476) Google Scholar, 29Zhou Y Fisher SJ Janatpour M Genbacev O Dejana E Wheelock M Damsky CH Human cytotrophoblasts adopt a vascular phenotype as they differentiate: a strategy for successful endovascular invasion?.J Clin Invest. 1997; 99: 2139-2151Crossref PubMed Scopus (810) Google Scholar Nuclei in these sections were visualized by staining with Hoechst 33342 (Molecular Probes, Eugene, OR). Briefly, tissues were fixed in 3% paraformaldehyde for 30 minutes, washed three times in phosphate-buffered saline (PBS), infiltrated with 5 to 15% sucrose followed by Optimal Cutting Temperature compound (Miles Scientific, Naperville, IL), and frozen in liquid nitrogen. Sections (6 μm) were prepared using a cryostat (Slee International Inc., Tiverton, RI) and collected on poly-L-lysine-coated microscope slides (Fisher Scientific, Pittsburgh, PA). Fixed sections were permeabilized in cold methanol for 5 minutes, washed three times in PBS for 5 minutes, and incubated for 30 minutes with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO) in PBS. Sections were then incubated for 1 hour with two primary antibodies (7D3 to detect trophoblasts and one that specifically reacted with a cell cycle regulator) followed by rinsing three times in PBS for 5 minutes. The sections were then incubated with secondary antibodies conjugated to rhodamine or fluorescein and washed three times in PBS for 5 minutes. Afterward, tissue sections were placed in the Hoechst dye (10 μg/ml PBS) for 2 minutes, washed in PBS (5 minutes), and mounted with Vectashield (Vector Laboratories, Burlingame, CA). All incubations and washes were performed at room temperature. Negative controls were included in every experiment. Either pre- or non-immune serum was always used for this purpose. Other controls included isotype-matched non-immune serum and omission of the primary antibody. In no instance was staining observed in the negative controls. Samples were examined with a Zeiss Axiophot epifluorescence microscope (Thornwood, NY) equipped with filters to selectively view the rhodamine and fluorescein fluorescence. Hoechst staining was photographed under ultraviolet illumination. Staining was evaluated as follows. In most cases trophoblast nuclei, as confirmed by Hoechst staining, either stained brightly or failed to react with an antibody. The only exception was anti-cyclin-D3, which showed faint reactivity. The fact that these antibodies strongly stained nuclei in human cell lines suggested that the patterns we observed were correct. Therefore, intensity of staining was not quantified. In contrast, there was a great deal of variation in the number of cells in different regions that reacted with all of the other antibodies. As a result the number of antibody-reactive cells was graded according to the following semiquantitative scale of percentage of CK-positive cells showing reactivity: +++, more than 75%; ++, 50 to 74%; +, 25 to 49%; +/−, 5 to 24%; −, less than 5%. Unless otherwise noted, the same results were obtained for samples of all gestational ages within a group. Starting with G1, we immunolocalized cell cycle regulators in tissue sections of the maternal-fetal interface that contained floating villi (which include CTBs differentiating into syncytium) or anchoring villi (which include CTBs differentiating into invasive cells, and often the site of uterine attachment). The tissue sections were double-stained with anti-cytokeratin, which specifically reacts with all populations of trophoblast cells. The results obtained from analysis of first and second trimester tissues are summarized in Table 1, Table 2, respectively. We focused on these stages because much placental development occurs during this period. Accordingly, proper coordination of CTB proliferation and differentiation during this time is a crucial determinant of pregnancy outcome.Table 1First Trimester Staining Patterns of Cell Cycle Regulators at the Fetal-Maternal InterfacePlacenta—fetal sideUterine wallCytotrophoblast cells (CTBs)Cell column CTBsInvasive CTBsCell cycle phaseMarker*No immunostaining was observed for cyclin D2, pRb, or unphosphorylated p53.SyncytiotrophoblastVillus CTBsProximalDistalSuperficialDeepG1Cyclin D1−+++++−−Cyclin D3†Cytoplasmic staining.−++++−−−CDK4−+++++−−−CDK6−+/−−−−−p16−−−+/−+++−G1-Sp21−+++++−p27−+++++++p57−+−+++++++P-p53†Cytoplasmic staining.−−+/−+/−−−Mdm2−+++++++−−SKi67−+++++++++−−Cyclin A++++++++++++++−G2-MCyclin B++++++++++++−Cdc2−++++++/−−−MHistone P-H3−+/−+/−++++−Immunostaining was scored on a semiquantitative scale as described in Materials and Methods. Results ranged from +++ (≥75% of cells stained) to − (≤5% of cells stained).* No immunostaining was observed for cyclin D2, pRb, or unphosphorylated p53.† Cytoplasmic staining. Open table in a new tab Table 2Second Trimester Staining Patterns of Cell Cycle Regulators at the Fetal-Maternal InterfacePlacenta—fetal sideUterine wallCytotrophoblast cells (CTBs)Cell column CTBsInvasive CTBsCell cycle phaseMarker*No immunostaining was observed for cyclin D2, pRb or unphosphorylated p53.SyncytiotrophoblastVillus CTBsProximalDistalSuperficialDeepG1Cyclin D1−++/−, −†+/−, 15 weeks; −, 20 weeks−−−Cyclin D3−−−−−−CDK4−+++−−−CDK6−+++−−−p16++++, +++‡++, 15 weeks; +++, 20 weeks+++++++++++G1-Sp21−+/−+/−−−−p27−+/−+++++++++p57−, ++§−, ≤15 weeks; ++, ≥20 weeks++++++++++++P-p53−−−−−−Mdm2−+++−−SKi67−+++−−−Cyclin A+++++++−G2-MCyclin B++++−−Cdc2−++++−−−MHistone P-H3−+/−+/−+/−+/−−Immunostaining was scored on a semiquantitative scale as described in Materials and Methods. Results ranged from +++ (≥75% of cells stained) to − (≤5% of cells stained).* No immunostaining was observed for cyclin D2, pRb or unphosphorylated p53.† +/−, 15 weeks; −, 20 weeks‡ ++, 15 weeks; +++, 20 weeks§ −, ≤15 weeks; ++, ≥20 weeks Open table in a new tab Immunostaining was scored on a semiquantitative scale as described in Materials and Methods. Results ranged from +++ (≥75% of cells stained) to − (≤5% of cells stained). Immunostaining was scored on a semiquantitative scale as described in Materials and Methods. Results ranged from +++ (≥75% of cells stained) to − (≤5% of cells stained). To begin mapping CTB progression through G1, we used antibodies to localize expression of D-type cyclins, together with their catalytic subunits, CDK4 and CDK6. The D-type cyclins serve as growth factor sensors that integrate extracellular signals with the cell cycle machinery. Together with their partner kinases, CDK4 and 6, they operate in early-to-mid G1 to promote progression through the G1-S restriction point. Thus their expression signals the cell’s commitment to replicate its genome.30Sherr CJ G1 phase progression: cycling on cue.Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 31Weinberg RA The retinoblastoma protein and cell cycle control.Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4277) Google Scholar Because these cyclin proteins have a very short half life (20 to 30 minutes), their protein levels are directly related to the rate of transcription.32Pines J Protein kinases and cell cycle control.Semin Cell Biol. 1994; 5: 399-408Crossref PubMed Scopus (93) Google Scholar Thus, staining patterns primarily identify cells in G1. Staining for cyclin D1 showed very specific expression patterns in both first and second trimester tissues. In first trimester samples most cells of the vCTB monolayer and about 25% of CTBs in cell columns (Figure 1A) reacted with the antibody. In both cases predominantly nuclear staining was observed. In the latter case patches of CTBs stained. Less frequently, cells in the villus core also reacted with anti-cyclin D1. The STB layer (Figure 1A) and CTBs in the uterine wall (data not shown) did not stain for cyclin D1. In second trimester samples, a much lower percentage of the vCTB monolayer stained. Sometimes a few stained cells in the proximal column regions were observed in very early second trimester samples (eg, 15 weeks of gestation). At 20 weeks, cells in the column no longer stained (Figure 1C). In the second trimester, we observed no staining of invasive CTBs within the uterine wall (data not shown) or of the syncytium (Figure 1C). To determine the degree of overlapping expression among the D-type cyclins, we also assessed the staining patterns of antibodies specific for cyclins D2 and D3, molecules that may play a role in cell proliferation or maintenance of terminal differentiation.33Bartkova J Lukas J Strauss M Bartek J Cyclin D3: requirement for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation.Oncogene. 1998; 17: 1027-1037Crossref PubMed Scopus (173) Google Scholar None of the trophoblast populations stained for cyclin D2, either in first or in second trimester samples (summarized in footnote to tables; data not shown). In first trimester tissue, anti-cyclin D3 failed to stain STBs, but did react with a substantial number of vCTBs and CTBs in the proximal region of cell columns. A cytoplasmic rather than a nuclear staining pattern was observed (data not shown). Because functionally active cyclins localize to the nucleus, the significance of this result was unclear. In second trimester tissue, anti-cyclin D3 did not stain any of the trophoblast populations (data not shown). We also examined the expression of CDK4, a catalytic partner of D-type cyclins. In first trimester samples, both nuclear and cytoplasmic staining were detected (Figure 1E), essentially in the same pattern as cyclin D1 expression; no staining for CDK4 was detected in CTBs past the mid-column region (data not shown). The same pattern was observed in second trimester samples, except that fewer vCTBs stained (data not shown). As to immunolocalization of CDK6, staining of first trimester samples was sometimes observed. Antibody reactivity was confined to a few vCTBs. In second trimester tissue CDK6 staining resembled that of CDK4 (data not shown); antibody-reactive cells were observed among the vCTB and proximal column populations. Next, we localized cyclin E expression. Levels of this cyclin peak at the G1-S transition, ie, later than the D-type cyclins. Cyclin E associates with CDK2. The peak of the associated kinase activity, which also occurs during late G1, is required for progression into S phase.34Heichman KA Roberts JM Rules to replicate by.Cell. 1994; 79: 557-562Abstract Full Text PDF PubMed Scopus (240) Google Scholar After a cell enters S phase, cyclin E is rapidly degraded, which frees CDK2 to associate with cyclin A. In general, we observed strong nuclear reactivity with anti-cyclin E in all CTBs (data not shown). Normally, cyclin E is expressed at the G1-S transition. However, we found cyclin E is coexpressed with cyclins that mark S and G2 (eg, cyclins A and D; see below). In light of this discrepancy, we could not interpret this staining pattern. To understand how CDKs promote cell cycle progression, it is important to identify their physiological substrates. Cyclin D-CDK4/6, cyclin E-CDK2, and cyclin A-CDK2 complexes have been implicated in sequential phosphorylation of the retinoblastoma protein (pRb). Phosphorylation of pRb releases E2F and other transcription factors that trigger the activation of S-phase (such as cyclin A35Bartek J Bartkova J Lukas J The retinoblastoma protein pathway and the restriction point.Curr Opin Cell Biol. 1996; 8: 805-814Crossref PubMed Scopus (363) Google Scholar) and regulates the restriction point of “start” for cell cycle progression.30Sherr CJ G1 phase progression: cycling on cue.Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 36Resnitzky D Reed SI Different roles for cyclins D1 and E in regulation of the G1-to-S transition.Mol Cell Biol. 1995; 15: 3463-3469Crossref PubMed Scopus (432) Google Scholar Staining of first and second trimester tissue samples for pRb was so weak that no obvious pattern could be discerned. Because commercially available antibodies do not discriminate between the phosphorylated (inactive) and nonphosphorylated (active) forms of pRb, this line of investigation was not pursued. In addition to cyclin binding, the activity of the G1 cyclin-CDKs is regulated by specific cyclin-dependent kinase inhibitors (CDKIs). CDKIs belong to two families that are differentiated by their targets. The INK4 family includes p15, p16, p18, and p19, which specifically inhibit the activity of CDK4/6 complexes.37Sherr CJ Roberts JM Inhibitors of mammalian G1 cyclin-dependent kinases.Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3200) Google Scholar In first trimester tissue anchoring villi, nuclear staining for p16, the most specific inhibitor of CDK4 and CDK6, was confined to a few CTBs in the distal region of cell columns (Figure 2A). Interestingly, most CTBs within the uterine wall expressed p16 (Figure 2C). In second trimester tissue, staining for p16 was significantly up-regulated in association with STBs, vCTBs (Figure 3A) and column CTBs, in both the proximal and the distal regions (Figure 3C). Most CTBs in all areas of the uterine wall stained for p16 (Figure 3C).Figure 3In second trimester samples, p16 was expressed by many trophoblasts in all locations. Sections of 15-week floating (A and B) and anchoring (C and D) villi and the portion o