Nelson K. S. Khoo

SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasivess and resistance to anti-invasive action of TGFβ

Invasion of the uterus by first trimester human placental extravillous trophoblast (EVT) cells depends on mechanisms shared by malignant cells. However, unlike tumor invasion, trophoblast invasion of the uterus is stringently controlled in situ by local molecules such as transforming growth factor (TGF)β. Since EVT cells possess active invasion‐associated genes but are nontumorigenic, our objective was to induce premalignant and then malignant phenotype into a normal EVT cell line in order to identify the molecular basis of tumor progression. Simian virus 40 large T antigen (SV40 Tag) was introduced into a normal human first trimester invasive EVT cell line, HTR8, established in our laboratory. Since the HTR8 line has a limited in vitro lifespan of 12–15 passages, SV40 Tag‐transformed cells were selected on the basis of extended lifespan. A long‐lived line, RSVT‐2, was produced and an immortalized subclone, RSVT2/C, was further derived under a forced crisis regimen. We examined transformation‐induced alterations in proliferative and invasive abilities, responses to the invasion and proliferation‐regulating growth factor TGFβ and changes in gene expression for invasion‐associated enzymes or enzyme inhibitors. RSVT‐2 and RSVT2/C cell lines were hyperproliferative and hyperinvasive when compared with the parental HTR8 cell line. They were also variably resistant to the anti‐proliferative and anti‐invasive signals from TGFβ. Since both cell lines remained non‐tumorigenic in nude mice, these properties indicate that they attained a premalignant phenotype. Both cell lines showed reduced expression of tissue inhibitor of metalloproteases (TIMP)‐1, while TIMP‐2 and plasminogen activator inhibitor (PAI)‐1 expression was was also reduced in RSVT2/C cells, thus contributing to their hyperinvasiveness. Their resistance to the anti‐invasive action of TGFβ was explained by the failure of TGFβ to upregulate TIMPs and PAI‐1, in contrast to the TGFβ‐induced upregulation noted in parental HTR8 cells. Int. J. Cancer 77:429–439, 1998.© 1998 Wiley‐Liss, Inc.

Immunotherapy of mammary adenocarcinoma metastases in C3H/HeN mice with chronic administration of cyclo-oxygenase inhibitors alone or in combination with IL-2.

In this study the efficacy of treatment of two cyclo-oxygenase inhibitors, ibuprofen (Ibu) and indomethacin (Indo), are compared in the immunotherapy of metastasis designed to reverse prostaglandin E2 (PGE2)mediated inactivation of interleukin-2 (IL-2)-dependent host killer cell lineages. These agents were tested either alone for the prevention of metastasis or in combination with IL-2 for the eradication of established metastasis. C3H/HeN mice were placed on chronic oral Ibu (CIbT; 200 and 600 ,μg/ml of water) or Indo (CIT; 10 μg/ml) 5 days after s.c. transplantation of 5 × 105 metastatic C3L5 mammary carcinoma for the prevention of spontaneous lung metastases. They showed intolerance to Indo at a dosage of 14 μg/ml, which was well tolerated by other mouse strains in previous studies, but tolerated the Ibu dosages used. Control and treated mice were killed on day 30 to score metastatic lung colonies, to evaluate killer activity in splenocytes against natural killer (NK)-sensitive YAC-1 lymphoma or NK-resistant C3L5 adenocarcinoma and 8911 lymphoma targets, and to phenotype the surface markers of killer cells. CIbT and CIT alone at the above dosage significantly reduced the number of lung colonies, retarded local tumor growth and restored NK activity of splenic killer cells expressing AGM-1+, Thy-1−, Lyt-2− phenotype. To treat established lung metastasis, mice bearing 15-day C3L5 transplants were given CIbT or CIT alone or in combination with two 4-day rounds (days 20–23, 31–34) of IL-2 (15 000 Cetus units, i.p. every 8 h) and were killed on day 35 to score lung colonies and characterize splenic killer cells. CIbT or CIT alone reduced the number of spontaneous lung metastases and restored anti-YAC-1 killer function of splenocytes with NK-like phenotype (AGM-1+, Thy-1−, Lyt-2−); some anti-C3L5 killer function was also generated in the high dose Ibu group and the killer cell showed AGM-1+, Thy-1+ and Lyt-2+ phenotype. Combined therapies with CIbT or CIT plus IL-2 were more effective in reducing metastases and promoting killer cell function, the best results being achieved with high dose Ibu + IL-2. All killer cells expressed AGM-1 and Thy-1. In addition, C3L5 killer cells also expressed Lyt-2, suggesting T-cell stimulation. PGE2 synthesis in the host was inhibited by at least 50% in mice subjected to CIbT or CIT. Thus, Ibu proved to be an excellent substitute for Indo in preventing metastasis and NK cell activation when given alone, and also in ameliorating established metastasis and activating lymphokine-activated killer cells when combined with IL-2.

SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. II. Changes in gap junctional intercellular communication

Poor gap junctional intercellular communication (GJIC) has been associated with uncontrolled cell growth and neoplasia. We have successfully propagated normal first trimester invasive extravillous trophoblast (EVT) cells, and have produced premalignant EVT lines after SV40 Tagtransformation: RSVT‐2 is an uncloned line that is long‐lived; RSVT2/C is a clonal line that is immortal. Both are hyperproliferative, hyperinvasive and variably refractory to the anti‐proliferative and anti‐invasive effects of transforming growth factor β (TGFβ). Possible changes in gap junctions during the transition of normal invasive EVT cells to the premalignant stage were examined by comparing expression of connexin proteins (by immunolabeling for Cx26, Cx32, Cx40, Cx43), and mRNA (by Northern blot with cDNA probes for Cx26, Cx32, Cx43), and functional GJIC (by dye transfer using the preloading method) in normal parental EVT cells and their SV40 Tag transformants. Results from immunofluorescence and Northern blot analysis revealed that, of the panel of connexins examined, only Cx43 was variably expressed in these cell lines in vitro. Expression of Cx43 protein and mRNA was abundant in normal EVT cell line HTR8, reduced in long‐lived RSVT‐2 cells and undetectable in immortalized RSVT2/C cells. GJIC, as measured by dye transfer between donor and recipient cells, was also similarly reduced in recipient RSVT‐2 cells, and drastically reduced in RSVT2/C cells, irrespective of whether the dye donor was of the same cell type (homocellular coupling) or HTR8 cells (heterocellular coupling). Treatment with TGFβ reduced Cx43 mRNA expression as well as GJIC in normal EVT cells, but not in the SV40 Tag transformants. Our findings suggest that downregulation of connexins with the resultant impairment in GJIC is an early event in tumor progression, as observed in the premalignant SV40 Tag transformants. Int. J. Cancer77:440–448, 1998.

Role of transforming growth factor-α (TGFα) and epidermal growth factor (EGF) on proliferation and invasion by first trimester human trophoblast

Summary By employing pure cultures of first trimester human trophoblast cells we have examined the effects of EGF and TGFα on trophoblast cell proliferation and invasiveness. Both exogenous EGF and TGFα were able to stimulate trophoblast proliferation ( 3 H-TdR incorporation). Anti-TGFα neutralizing Ab had no effect on proliferation indicating the lack of significant endogenous TGFα production in these cultures. Anti-EGF-receptor blocking Ab significantly decreased trophoblast proliferation indicating the presence of endogenous EGF or another EGF-receptor ligand, capable of binding to the EGF-receptor, and enhancing trophoblast growth. EGF and TGFα did not alter trophoblast invasiveness in a Matrigel invasion assay although an increase in the transcription of invasion regulating molecules, type IV collagenases and their inhibitors TIMP1 and TIMP2 was noted. Both growth factors may be required for normal placental growth in situ . These studies reveal that the two important biological processes, e.g., trophoblast proliferation and invasion required for placental development are not necessarily linked.

Control mechanisms in human trophoblast proliferation and invasiveness and their derangement during trophoblastic tumor progression

Summary Extravillous trophoblast (EVT) cells of the human placenta proliferate, migrate and invade the decidua and its vasculature. By utilizing in vitro propagated normal first trimester human EVT cells in functional assays, we have shown that proliferative, migratory and invasive functions of these cells are stringently regulated in situ by numerous growth factors, their binding proteins and proteoglycans and extracellular matrix (ECM) components. Growth factors in the EGF family (EGF, TGFβ and amphiregulin), CSF-1, VEGF and PIGF all stimulate EVT cell proliferation without affecting migratory or invasive abilities, whereas IGF-II and its binding protein IGFBP-1 stimulate EVT cell migration and invasiveness without affecting proliferation. Finally, TGFβ a major decidual cell product, inhibits proliferation, migration and invasiveness of normal EVT cells, whereas choriocarcinoma cells defy the TGFβ mediated control. We have produced “premalignant” derivatives of normal EVT cells by SV40 Tag transfection, some of which are immortal (as opposed to normal EVT cells which senesce at 5–15 passages), hyperproliferative, hyperinvasive, resistant to antiproliferative and antiinvasive action of TGFβ and deficient in gap junctional intercellular communication, but are yet incapable of anchorage independent growth or tumorigenicity in nude mice. Transition of the normal EVT cell to the premalignant stage is associated with multiple genetic changes, e.g., a downregulation of connexins, TIMP-1, TIMP-2 and PAI-I, which partially account for the phenotypic changes. We have recently intruduced activated H-ras oncogene into a premalignant EVT cell line to induce malignant/metastatic phenotype. Normal, premalignant and malignant EVT cell lines derived from a single placenta provide us with an exquisite in vitro model for studies of stage-specific genetic changes responsible for trophoblastic tumor progression.

Effects of histamine type-2 receptor antagonists on indomethacin and IL-2 immunotherapy of metastasis

Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 × 105 C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 µg/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 µg/ml (Ran-lo) or 143 µg/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 µg/ml, or (c) cimetidine (Cim) at 107 µg/ml, all in the drinking water on days 5–24; (3) IL-2 (1.5 × 103 Cetus U i.p. every 8 h on days 10–14 and 20–24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (51Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no differences were observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy.

Regulation of NM23 gene expression in the normal and malignant trophoblast by growth factors

Summary nm23-H1 is a nucleoside diphosphate kinase with a functional role in the microtubular assembly and disassembly during cell proliferation and in G-protein dependent signal transduction. Gene expression for this kinase has been reported to be inversely correlated with metastatic ability of tumor cells. Normal trophoblast cells are highly invasive but non-metastatic. Numerous growth factors, e.g., TGF-α, EGF, TGF-β, and CSF-1, present at the fetomaternal interface, influence trophoblast functions such as proliferation, differentiation, and invasion. In this study, we investigated the influence of the above growth factors on nm23-H1 mRNA expression by first trimester normal trophoblast and two choriocarcinoma cell lines, JAr and JEG-3. The expression was found to be abundant in the normal trophoblast as well as choriocarcinomas, indicating that this expression was not suppressed in malignant trophoblast cells. We found that growth factors (e.g., TGFα, EGF, and CSF-1) which stirnulate normal trophoblast proliferation also stimulated nm23-H1 expression in the normal trophoblast indicating that the expression may be coupled with cell proliferation. A stimulatory effect noted with a neutralizing anti-TGF-β antibody can also be explained on the basis of stimulation of trophoblast proliferation due to removal of the anti-proliferative action of endogenous TGF-β. The expression of nm23H-1 in choriocarcinoma cells was unaltered by the growth factors except for some stimulation of JEG-3 cells noted with TGF-β. Since anti-TGF-β Ab also stimulated the expression in JEG-3 cells, we propose that TGF-β may have a dual influence in this case, one related to signal transduction, another to its anti-proliferation.