—  SYMPOSIUM #41  —

Gestational Trophoblastic Disease
Moderator: Dr. le-Ming Shih

Section 2 - Cellular and Molecular Mechanisms in Trophoblast Invasion and Tumor Progression*
 *Supported by a grant from the Canadian Institutes of Health Research

P. K. Lala
Dept. of Anatomy & Cell Biology
University of Western Ontario
London , Ontario , CANADA


Introduction
The "hemochorial" type of placenta in a number of species including the human is a highly invasive "pseudo-tumor"-like structure that invades the pregnant uterus and uteroplacental arteries to tap on the maternal blood supply and nourish the fetus. This phenomenon poses two important biological riddles in nature:
  1. What protects the fetally-derived placenta, which can be considered as an allograft (carrying both paternal and maternal histocompatibility genes) in the mother's uterus, from maternal immune attack?
  2. What limits the invasion of the pregnant uterus by the invasive cells of the placenta, so that the uterus remains unharmed?
Our laboratory has contributed to the understanding of both these riddles, and the present talk will focus on the second riddle. I shall summarize studies from our laboratory to address two important issues:
  1. What are the molecular mechanisms regulating placental invasiveness in order to maintain a healthy utero-placental homeostasis?

  2. What are the molecular/genetic changes associated with a loss of control of placental invasiveness during trophoblastic tumor progression?

Cell responsible for placental invasiveness: the extravillous trophoblast. Dr. Shih has already presented an overview of the various trophoblastic subpopulations belonging to the normal placenta. I shall focus on the population termed as the "intermediate trophoblast" (IT) by the pathologists, also named as the "extravillous trophoblast" (EVT) by the placentologists. This population arises by proliferation and differentiation of cytotrophoblast "stem cells" in certain chorionic villi (anchoring villi), wherefrom they migrate as cell columns to invade the decidua and the uteroplacental arteries. At the base of the columns near the villous tips wherefrom they emerge, EVT cells are highly proliferative; however, their proliferative activity is lost progressively as they approach the decidua with a concomitant increase in cellular ploidy, and a complete loss of proliferation within the decidua [1]. Within the implantation site decidua they present as several subsets [2, 3]: (a) interstitial trophoblast dispersed within the decidua; (b) endovascular trophoblast which invade spiral arteries in the endometrium and myometrium, modifying them into noncontractile tubes allowing a steady flow of maternal blood into the sinusoids. These cells acquire an endothelial phenotype (expressing VCAM-1 and ephrin B receptor EPHB4) and replace the endothelium of these arteries [4]; (c) placental bed giant cells which are non-invasive, arise by EVT cell fusion and make hPL; (d) a continuous layer of cytotrophoblastic shell separating the decidua from the placental sinusoids.

Molecular mechanisms responsible for EVT invasiveness. We have succeeded in propagating normal first trimester invasive human EVT cells from chorionic villus explant cultures [5, 6] to show that they have all the phenotypic properties of the EVT cells in situ [6], expressing cytokeratin-7, EGF-R, placental type alkaline phosphatase, receptor for urokinase type plasminogen activator (uPA), HLA framework antigen, and integrin (receptors for extracellular matrix, ECM) subunits a1, a3, a5, av, β1 and vitronectin receptor, but not a 6 or β4 subunits expressed by the villous trophoblast. They express HLA-G when grown on laminin or matrigel (reconstituted ECM) [7]. Using in vitro invasion assays with natural or reconstituted basement membrane substrates [3, 8, 9, 10], we found that their invasiveness depends on numerous steps: (a) binding to ECM components laminin, collagen type IV and fibronectin, (b) degradation of the ECM by production of matrix metalloproteases (MMP-2, MMP-9 and MT-1 MMP) and uPA (which activates MMPs), and (c) migration through the degraded ECM. Their migration requires the expression of Asn-linked complex type oligosaccharides [11] as well as binding to fibronectin via a5β1 integrin on the EVT cell surface [12]. The balance between the production of MMPs and their inhibitors (tissue inhibitors of metalloproteases, TIMPs; particularly TIMP-1), as well as uPA and its inhibitor (PAI-I), dictates their matrix-degrading ability [2, 3, 9]. While these mechanisms are identical to those of invasive and metastatic tumor cells [2, 8, 9], normal invasive EVT propagated in culture are neither tumorigenic nor metastatic in nude mice, and they have a limited life span (5-15 passages) [6, 13], indicating that invasiveness is an essential but not sufficient pre-requisite for tumorigenicity or metastasis, and that EVT invasion in situ must be controlled locally.

Molecular mechanisms regulating trophoblast growth, migration and invasion in situ. We discovered that the regulation is provided by the interaction of the EVT with certain locally derived growth factors, growth factor binding proteins, proteoglycans and ECM components for which EVT cells have receptors. Factors blocking EVT cell proliferation, migration and invasiveness: Two decidua-derived factors, transforming growth factor (TGF)-β and a TGFβ-binding proteoglycan decorin that localizes TGFβ in the decidual ECM, inhibit EVT cell growth, migration and invasiveness independently of each other [14, 15, 16, 17]. We suggest that decorin serves as a storage device for inactive TGFβ which is released from the TGFβ-decorin complex and activated by EVT cell derived proteases, triggering negative control against trophoblast over-invasion of the decidua [17]. TGFβ negatively regulates EVT proliferation as in other epithelial cells [14] and blocks EVT invasion by (a) upregulating TIMP-1 mRNA and protein which neutralizes MMPs [15], (b) down-regulating uPA [13], (c) upregulating PAI-I [16] and (d) reducing their migratory ability, apparently by upregulating integrins [12], making the cells more adhesive to the ECM. The mechanisms for negative regulatory functions of decorin on EVT cells remain to be fully identified. Interestingly, choriocarcinoma cells are refractory to both TGFβ and decorin-mediated negative regulation. Factors stimulating EVT cell growth: All EGF receptor ligands (EGF, TGF-a, amphiregulin), CSF-1, VEGF and PlGF produced locally by the trophoblast and/or the decidua stimulate EVT cell proliferation, having little effect on invasion [18, 19, 20, 21, 22, 23]. Migration/Invasion promoting factors: These factors are numerous. IGF-II (produced by the EVT) stimulates their migration [12] and invasiveness without influencing proliferation [24] by signaling through the type II IGF receptor (IGF-RII), inhibition of adenylyl cyclase and stimulation of MAP kinase (MAPK) [25]. An IGF binding protein, IGFBP-1, released by decidual cells also promotes invasion [24], due to stimulation of EVT migration [12] because of binding of its RGD (arginine-glycine-aspartic acid) sequence to the a5β1 integrin on the EVT cell surface, followed by activation of focal adhesion Kinase (FAK) and MAPK [26]. EVT cell-derived urokinase type plaminogen activator (uPA) exerts dual functions: promotion of invasiveness by its catalytic domain, and promotion of migration by binding to uPA receptors on the EVT cell surface [27]. Indeed, derangements in the production or function of some of these migration/invasion-promoting factors e.g. uPA/uPAR system or IGFBP-1 protein may be instrumental in the development of preeclampsia, a trophoblastic hypoinvasive disorder [28]. For example, reduced IGFBP-1 level in maternal serum (apparently resulting from a low production by the decidua) during early pregnancy (15-16 weeks gestation) may be a predictive marker for the development of this disease before the appearance of clinical signs [28]. Other molecules promoting EVT cell migration are endothelin (ET)-1 made by many cells in the placenta or the decidua [29] and prostaglandin E2 (PGE2) made by the decidua. In the latter case, PGE2 receptor class EP1 appears to play a major role [30].

Proliferative and invasive functions of EVT cells are not mutually exclusive. Invasive function of EVT cells does not necessarily indicate a terminal differentiation into a non-proliferative state. By using primary cultures of EVT cells in invasion chambers containing matrigel and collecting those that have invaded matrigel, we discovered that even after the act of invasion, EVT cells retained significant, but limited proliferative ability [17]. This finding suggests that progressive loss of proliferative ability of EVT cells in situ as they migrate to the decidua result from a dual mechanism: endoreduplication without mitosis [1], and proliferation inhibition by decidua-derived anti-proliferative factors, e.g. TGFβ and decorin [17].

Molecular/Genetic Mechanisms in Trophoblastic Tumor Progression
To identify molecular and genetic mechanisms leading to specific stages in trophoblastic tumor progression, we utilized normal, premalignant and malignant human EVT (choriocarcinoma) cells as a model system in vitro. That choriocarcinomas are malignant derivatives of EVT cells is suggested by the fact that the EVT cell specific marker HLA-G was originally cloned from JEG-3 choriocarcinoma cells [31]. Since premalignant EVT cell lines have not been produced from in situ lesions, we derived them in our laboratory by genetic manipulation.

Derivation of premalignant EVT cells. We stably transfected two different SV40 Tag-containing plasmids into a normal short-lived human first trimester EVT cell line, HTR-8. Transfection with pSV3neoSV40 Tag having a selectable marker for neomycin resistance resulted in the HTR-8/SVneo cell line which has identical phenotype as HTR-8 cells except for immortality (living beyond 100 passages). It is responsive to antiproliferative [13], antiinvasive and antimigratory [26] as well as fibronectin-upregulatory signals of TGFβ, lacks in agar colony forming ability or tumorigenicity in nude mice. Thus it is neither premalignant nor malignant, indicating that SV40 Tag transfection per se did not induce premalignancy or malignancy. The second line RSVT2/C [32] was produced by transfection with the non-selectable pRSV-Tag and selected on the basis of prolonged longevity and derivation of an immortalized clone after a forced crisis by serum starvation in culture. RSVT 2/C cells show higher proliferative and invasive abilities, and resistance to anti-proliferative, anti-invasive and fibronectin-up regulatory actions of TGFβ. However, unlike choriocarcinoma cells, they are unable to form agar colonies or tumors in nude mice. A dramatic decline in TIMP-1, TIMP-2 and PAI-I mRNA in RSVT 2/C cells may explain their increased invasiveness [32]. Finally, gap-junctional intercellular communication (GJIC) as well as the expression of connexin-43 mRNA and protein noted in parental HTR-8 cells were undetectable in RSVT 2/C cells [33]. This is a hallmark of carcinogenesis [34]. These characteristics, taken together, reveal that RSVT 2/C cells have attained a premalignant phenotype.

Using differential display (DD) of reverse-transcribed mRNA, we identified a total of 18 different gene sequences with differential expression between the normal HTR-8 and the premalignant RSVT2/C cells [35]. Of these sequences, 7 (up or down-regulated) genes are of unknown identity, while the remainder include potential tumor suppressors such as insulin-like growth factor binding protein (IGFBP)-5 and fibronectin (hFN) as well as genes with oncogenic potential such as Chromokinesin (KIF4), alternative splicing factor (SF) 2, dynein, human DNA polymerase ε subunit p12 and NF-κB-activating kinase (NAK). The function of the remaining 4 genes FK506 binding protein (FKBP5), histone protein (HP1Hs)-γ, nucleoporin (Nup) 155 and an 82kDa acidic human protein, which are upregulated in the premalignant EVT, remains unknown. We found that loss of IGFBP-5 in conjunction with the loss of type 2 IGF receptor (1GF-IIR) in the premalignant RSVT2/C cells has emancipated them from the negative regulatory control of IGFBP-5 on IGF-1 dependent proliferation of these cells. Loss of IGF-IIR, a recognized tumor-suppressor gene was unique to the premalignant EVT, unrelated to Tag transfection alone, since this was not seen in HTR-8/SVneo cells [35].

TGFβ is the key factor controlling EVT cell growth, migration and invasiveness in situ [3], whereas premalignant EVT [32] as well as malignant EVT i.e. choriocarcinoma cells [36] are resistant to negative regulation by TGFβ. As a first step in identifying TGFβ signaling defect(s) responsible for TGFβ resistance in premalignant and malignant EVT, we compared the expression of TGFβ signaling molecules in the normal (HTR-8), premalignant (RSVT2/C) and malignant (JAR and JEG-3 choriocarcinoma) EVT cells [37]. RT-PCR analysis revealed that all these cell lines expressed the mRNA for TGFβ1, β2 and β3, TGFβ receptors I, II and III and post-receptor signaling genes Smad 2, 3, 4, 6 and 7 with the exception that TGFβ 2 and Smad3 were undetectable in choriocarcinomas. Smad 3 loss in the latter was verified at the protein level in immunoblots. TGFβ -induced Smad3 phosphorylation and translocation to the nucleus was demonstrable in the normal and premalignant, but not in malignant EVT cells [37]. Thus, Smad 3 disruption, which has not been reported in any other cancer, constitutes at least one of the TGFβ signaling defect(s) in choriocarcinomas that may account for TGFβ resistance. Indeed, we found that introduction of dominant negative Smad3 into normal EVT (HTR8/SVneo) cells made them partially resistant to growth inhibitory effects of "nodal", another member of the TGFβ superfamily [38].

To test whether TGFβ response can be restored by restitution of Smad3 in JAR cells we made stable Smad3-transfected JAR cell clones [39]. Since anti-invasive effects of TGFβ in the normal EVT are combined results of upregulation of PAI-1 [16], a down regulation of uPA [13, 15] and a down regulation of TIMP-1 [15], we tested the above TGFβ responses in one of the Smad 3 restituted clones JAR Smad 3c [39, 40]. This clone exhibited further upregulation of Smad 3 in the presence of TGFβ 1. The basal levels of PAI-1 mRNA and protein as well as uPA proteins were found to be very low in JAR and JAR-Smad3c cells, as compared to normal EVT cells. However, TGFβ1 significantly upregulated PAI-1 and downregulated uPA in JAR-Smad3/c but not in wild type JAR cells [39]. Similarly, the expression of TIMP-1 mRNA was found to be low in JAR as well as JAR-Smad3c cells as compared to normal EVT cells [40]. Exposure to TGFβ 1 upregulated TIMP-1 mRNA and secreted protein in Smad3 restituted JAR as well as in normal EVT cells but not in wild type JAR cells. However, in vitro functional assays revealed that in spite of significant restoration of uPA/PAI-1 as well as TIMP-1 responses to TGFβ , Smad3 restitution was insufficient in full restoration of antiinvasive function of TGFβ [40]. Thus additional factors (possibly low Smad4 expression and/or other unidentified defects downstream of Smad or in Smad-independent signaling) may also contribute to TGFβ unresponsiveness in JAR cells. These possibilities are currently being explored.

Conclusions
Extravillous trophoblast cells responsible for the invasive function of the placenta utilize similar molecular mechanisms for their invasiveness as cancer cells, however, unlike cancer cells, their proliferative, migratory and invasive functions are stringently controlled in situ by certain decidua-derived factors, in particular, TGFβ. Progression of normal EVT cells to a premalignant state is associated with changes in the expression of many genes some of which can explain their premalignant behavior. Loss of anti-invasive effects of TGFβ on the premalignant EVT is explained by down-regulation of TIMP-1, TIMP-2, and PAI-1 genes. Loss of TGFβ sensitivity in the malignant EVT (choriocarcinoma) is partially explained by the loss of expression of a transcription factor Smad3. Whether genetic changes noted in our premalignant EVT cell lines occur in situ remain to be investigated [41]. Availability of cell lines derived from in situ lesions representing various steps in trophoblastic tumorigenesis [42] should help future studies in this field.

References
  1. Zybina TG, Frank HG, Biesterfeld S, Kaufmann P. Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. II. Mechanisms of polyploidization. Tsitologiia. 2004;46(7):640-8.

  2. Graham, C.H. and Lala, P.K. (1992) Mechanisms of placental invasion of the uterus and their control. Biochem. Cell Biol. 70, 867, 874.

  3. Lala, P.K. and Hamilton , G.S. (1996) Growth factors, proteases and protease inhibitors in the maternal-fetal dialogue. Placenta 17, 545-555.

  4. Red-Horse K, Kapidzic M, Zhou Y, Feng KT, Singh H, Fisher SJ. EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation. Development. 2005 Sep;132(18):4097-106. Epub 2005 Aug 17.

  5. Yagel, S., Casper , R.F., Powell, W., Parhar, R.S. and Lala, P.K. (1989) Characterization of pure human first-trimester cytotrophoblast cells in long-term culture: growth pattern, markers and hormone production. Amer. J. Obstet. Gynecol. 160, 938-945.

  6. Irving , J.A., Lysiak, J.J., Graham, C.G., Hearns, S., Han, V.K.M. and Lala, P.K. (1995) Characterization of trophoblast cells migrating from first trimester human chorionic villus explants propagated in culture. Placenta 16, 413-433.

  7. ZdravKovic, M., Aboagye-Mathiesen, G., Guimond, M.-J., Hager, H., Ebbesen, P. and Lala, P.K. (1999) Susceptibility of MHC Class 1 expressing extravillous trophoblast cell lines to killing by natural killer cells. Placenta 20, 431-440.

  8. Yagel, S., Parhar, R.S., Jeffrey, J.J. and Lala, P.K. (1988) Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells. J. Cell. Physiol. 136, 455-462.

  9. Lala, P.K. and Graham, C.G. (1990) Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metastasis Rev. 9, 369-379.

  10. Graham, C.H., McCrae, K.R. and Lala, P.K. (1992) Molecular mechanisms controlling trophoblast invasion of the uterus. Troph. Res. 7, 237-250.

  11. Yagel, S., Kerbel, R.S., Lala, P.K., Eldar-Geva, T. and Dennis, J.W. (1990) Basement membrane invasion by first trimester human trophoblast: requirements for branched complex-type Asn-linked oligosaccharides. Clin. Exp. Metastasis 8, 305-317.

  12. Irving , J.A. and Lala, P.K. (1995) Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGFβ, IGF-II and IGFBP-1. Exp. Cell Res. 217, 419-427.

  13. Graham, C.H., Hawley, T.S., Hawley, R.G., MacDougall, J.R, Kerbel, R.S., Khoo, N. and Lala, P.K. (1993) Establishment and characterization of first trimester human trophoblast cells with extended life span. Exp. Cell Res. 206, 204-211.

  14. Graham, C.H., Lysiak, J.J., McCrae, K.R. and Lala, P.K. (1992) Localization of transforming growth factor-β at the human fetal-maternal interface: role in trophoblast growth and differentiation. Biol. Reprod. 46, 561-572.

  15. Graham, C.H. and Lala, P.K. (1991) Mechanism of control of trophoblast invasion in situ. J. Cell Physiol. 148, 228-234.

  16. Graham, C. H. (1997) Effect of transforming growth factor-beta on the plasminogen activator system in cultured first trimester human cytotrophoblasts. Placenta.18(2-3),137-43.

  17. Xu, G., Guimond, M.-J., Chakraborty, G. and Lala, P. K. Control of proliferation, migration and invasiveness of human extravillous trophoblast by decorin, a decidual product. Biology of Reproduction 67, 681-689, 2002.

  18. Lysiak, J.J., Han, V.K.M. and Lala, P.K. (1993) Localization of transforming growth factor-a (TGF-a) in the human placenta and decidua: Role in trophoblast growth. Biol. Reprod. 49, 885-894.

  19. Lysiak, J.J., Connelly, I. , Khoo, N.K., Stetler-Stevenson, W. and Lala, P.K. (1994) Role of transforming growth factor (TGF)a and epidermal growth factor (EGF) on proliferation and invasion by first trimester human trophoblast. Troph. Res. 8, 455-467.

  20. Lysiak, J.J., Johnson, G.R. and Lala, P.K. (1995) Localization of amphiregulin in the human placenta and decidua throughout gestation: Role in trophoblast growth. Placenta 16, 359-366.

  21. Hamilton , G.S., Lysiak, J.J., Watson, A.J. and Lala, P.K. (1998) Effects of colony stimulating factor (CSF)-1 on human extravillous trophoblast growth and invasion. J. Endocronol. 159, 69-77.

  22. Athanassiades, A., Hamilton , G.S. and Lala, P.K. (1998) Vascular endothelial growth factor stimulates proliferation but not invasiveness in human extravillous trophoblast. Biol. Reprod. 59, 643-654.

  23. Athanassiades, A. and Lala, P.K. (1998) Role of placental growth factor (PIGF) in human extravillous trophoblast proliferation, migration and invasiveness. Placenta 19, 465-473.

  24. Hamilton , G.S., Lysiak, J.J., Han, V.K.M. and Lala, P.K. (1998) Autocrine-paracrine regulation of human trophoblast invasiveness by insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-1. Exp. Cell Res. 244, 147-156.

  25. McKinnon, T., Chakraborty, C., Gleeson, L., Chidiac, D. and Lala, P.K. (2001) Stimulation of human extravillous trophoblast (EVT) migration by IGF-II is mediated by IGF type 2 receptor, involving inhibiting G proteins and phosphorylation of MAPK. J. Clin. Endocrinol. Metab. 86, 3665-3674.

  26. Gleeson, L., Chakraborty, C.,McKinnon, T. and Lala, P.K. (2001) Insulin-like growth factor binding protein-1 stimulates human trophoblast migration by signalling through a5β1 integrin via mitogen-activated protein Kinase pathway. J. Clin. Endocrinol. Metab.86, 2484-2493.

  27. Liu, J., Chakraborty, C., Barbin Y.P., Dixon S.J. and Lala P.K. Non catalytic domain of uPA stimulates human extravillous trophoblast migration by utilising phospholipase C, P-I-3 kinase and MAP kinase, Exp. Cell Res. 286, 138-151, 2003.

  28. Lala, P.K. and Chakraborty C. Factors regulating trophoblast migration and invasiveness: Possible derangements contributing to preeclampsia and fetal injury (current topic). Placenta, 24 (6) 575-587, 2003, 2003.

  29. Chakraborty, C., Barbin Y.P., Dixon , S.J., Chakraborty, S., Chidiac P. and Lala, P.K. Endothelin-1 promotes migration, elevation of [Ca++] i and phosphorylation of MAP kinase in a human extravillous trophoblast cell line. Mol. Cell Endcrinol. 201, 63-73, 2003.

  30. Nicola C., Timoshenko A.V., Dixon J., Lala P.K. and Chakraborty C. EP1 receptor-mediated migration of the first trimester human extravillous trophoblast: the role of intracellular calcium and calpain. J Clin Endocrinol Metab. 90(8), 4736-46, 2005.

  31. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ and DeMars R. (1990) A class I antigen, HLA-G, expressed in human trophoblasts. Science. 248, 220-3.

  32. Khoo, N.K.S., Bechberger, J.F., Shepherd, T., Bond, S.L., McCrae, K., Hamilton , G.S. and Lala, P.K. (1998) SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasivness and resistance to antiinvasive action of TGFβ. Int. J. Cancer 77, 429-439.

  33. Khoo, N.K.S., Zhan, Y., Bechberger, J.F., Bond, S.L., Hum, K. and Lala, P.K. (1998) SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. II. Changes in gap junctional intercellular communication Int. J. Cancer 77, 440-448.

  34. Yamasaki, H. (1990) Gap junctional intercellular communication and carcinogenesis. Carcinogenesis. 11, 1051-1058. Review.

  35. Lee BP, Rushlow WJ, Chakraborty C, and Lala PK. (2001) Differential gene expression in premalignant human trophoblast: role of IGFBP-5. Int J Cancer.94, 674-684.

  36. Graham, C.H., Connelly, I.H., MacDougall, J.R., Kerbel, R.S., Stetler-Stevenson, W.G. and Lala, P.K. (1994) Resistance of malignant trophoblast cells to both anti-proliferative and anti-invasive effects of transforming growth factor β. Exp. Cell Res. 214, 93-99.

  37. Xu, G., Chakraborty, C. and Lala, P. K. (2001) Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells. Biochem Biophys Res Commun. 287, 47-55.

  38. Munir, S, Xu, G., Wu, Y., Yang, B., Lala P.K. and Peng C. Nodal and activin receptor-like kinase (ALK) 7 inhibits proliferation and induce apoptosis in human trophoblast cells. J Biol Chem. 279 (30) 31277-31286, 2004.

  39. Xu G, Chakraborty C and Lala PK. Restoration of TGF-beta regulation of plasminogen activator inhibitor-1 in Smad3-restituted human choriocarcinoma cells. Biochem Biophys Res Commun. 294, 1079-86, 2002.

  40. Xu, G., Chakraborty, C. andLala, P. K. Reconstitution of Smad3 restores TGF- b response of tissue inhibitor of metalloprotease-1 upregulation in human choriocarcinoma cells. Biochem Biophys Res Commun. 300, 383-390, 2003.

  41. Lala PK, Lee BP, Xu G, and Chakraborty C. (2002) Human placental trophoblast as an in vitro model for tumor progression. Can J Physiol Pharmacol.80, 142-149.

  42. Shih, I-M.and Kurman, R.J. (1998) Ki-67 labeling index in the differential diagnosis of exaggerated placental site, placental site trophoblastic tumor, and choriocarcinoma: a double immunohistochemical staining technique using Ki-67 and Mel-CAM antibodies. Hum. Pathol. 29, 27-33.

  43. Massague J, Chen YG. Controlling TGF-beta signaling. Genes Dev. 2000 Mar 15;14(6):627-44. Review.