PIBF1 suppresses the ATR/CHK1 signaling pathway and promotes proliferation and motility of triple-negative breast cancer cells.

PURPOSE: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options. METHODS: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice. RESULTS: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line. CONCLUSION: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.

High Immune Expression of Progesterone-Induced Blocking Factor in Epithelial Ovarian Cancer.

BACKGROUND: Progesterone-induced blocking factor, which is released from maternal lymphocytes during pregnancy mediates the immune effect of progesterone. According to new reports, it is suggested that proliferating cells, such as human trophoblasts, mesenchymal stem cells, and malignant tumors, can excrete progesterone-induced blocking factor at high ratio to escape from maternal immunity. It is shown in recent studies that progesterone-induced blocking factor is overexpressed in many malignant tumors such as breast, cervical, lymphoma, and leukemia. There are no data about progesterone-induced blocking factor expression in ovarian cancer cells. Hence, it is aimed to determine the progesterone-induced blocking factor expression levels in epithelial ovarian cancer. METHODS: The study which was a retrospective cross-sectional study was conducted in a University Hospital. Twenty tissue specimens of patients with epithelial ovarian cancer and 20 tissue specimens of patients with healthy ovary were included in the study. Primary rabbit polyclonal anti- progesterone-induced blocking factor antibody was used to incubate the sections at a ratio of 1:300. RESULTS: When the tissue sections were compared based on immunostaining with progesterone-induced blocking factor, we detected high stromal progesterone-induced blocking factor expression in the epithelial ovarian cancer group as check against to the normal ovarian group ( P = .007). Similarly, we found high glandular progesterone-induced blocking factor expression in the epithelial ovarian cancer group as check against to the normal ovarian group ( P < .001). CONCLUSION: Proving the existence of progesterone-induced blocking factor expression in epithelial ovarian cancer cells may lead new visions or new studies for epithelial ovarian cancer immunotherapy. As a result, epithelial ovarian cancer cells have greater levels of expression of progesterone-induced blocking factor protein than normal ovarian tissue according to immunohistochemistry. Further research is needed to understand the clinical importance of this finding, to learn outcomes of high levels of progesterone-induced blocking factor, and to investigate its underlying mechanisms.

Expression of kisspeptin/GPR54 and PIBF/PR in the first trimester trophoblast and decidua of women with recurrent spontaneous abortion.

Kisspeptin and its receptor GPR54 play a major role in trophoblast invasion, and progesterone-induced blocking factor (PIBF) is needed for maintaining pregnancy. The expression of kisspeptin/GPR54 and PIBF/progesterone receptor (PR) in trophoblasts and deciduas and the relationship between kisspeptin and PIBF were investigated in the same women with recurrent spontaneous abortion (RSA). Trophoblastic and decidual tissues were collected from 32 RSA women who miscarried a genetically normal fetus, and 35 women who had voluntary abortion. Kisspeptin, GPR54, PIBF and PR were investigated using immunohistochemistry. Kisspeptin, GPR54 and PIBF expressions in syncytiotrophoblasts and cytotrophoblasts were decreased in RSA women as compared to controls (P<0.05). Kisspeptin, PIBF and PR expressions in deciduas were significantly decreased in RSA women as compared to controls (P<0.01). GPR54 expression in deciduas nearly showed no difference between the RSA group and the control group (P=0.958). Kisspeptin and PIBF expressions in syncytiotrophoblasts, cytotrophoblasts and deciduas were correlated with each other in the RSA group (Kappa=0.602, P=0.001; Kappa=0.590, P=0.001; Kappa=0.392, P=0.011). These data support the hypothesis that decreased kisspeptin and PIBF expressions in trophoblasts and deciduas are associated with RSA.

Dydrogesterone supplementation in women with threatened preterm delivery--the impact on cytokine profile, hormone profile, and progesterone-induced blocking factor.

Progesterone is indispensable in creating a suitable endometrial environment for implantation, and also for the maintenance of pregnancy. Successful pregnancy depends on an appropriate maternal immune response to the fetus. A protein called progesterone-induced blocking factor (PIBF) acts by inducing Th2-dominant cytokine production to mediate the immunological effects of progesterone. The aim of this prospective study was to compare serum concentrations of progesterone (P), estradiol (E2), anti-inflammatory (IL-10) and pro-inflammatory (IL-6, TNFα, IFNγ) cytokines, and serum PIBF concentrations in women with threatened preterm delivery who were given progesterone supplementation (study group) with those of women with threatened preterm delivery who were not given progesterone supplementation (control group). After dydrogesterone treatment of patients in the study group, serum PIBF as well as progesterone concentrations significantly increased. Women in this group had significantly higher serum levels of IL-10 than controls. The length of gestation was significantly higher in the group of women who were given progesterone supplementation. Our data suggest that dydrogesterone treatment of women at risk of preterm delivery results in increased PIBF production and IL-10 concentrations, and lower concentrations of IFNγ.

New target cells of the immunomodulatory effects of progesterone.

It is well known that the reproductive steroid hormones, particularly progesterone, in addition to its widely recognized effects on endometrial epithelial and stromal cells and spiral arteries, affect the activities of T cells and natural killer cells in the deciduas, thus inducing active immune tolerance against the fetal antigens. The immunomodulatory effects of progesterone on T cells, B cells and natural killer cells have been discussed extensively in the literature. The aim of the present review is to sum up and discuss the results from this and other laboratories of investigations on the effects of progesterone on dendritic cells and adult stem cells, which are some of the other cell populations present at the fetal-maternal interface and possibly are related to the immunoregulation during pregnancy. These cells have been shown to have a number of specific functions but their involvement in the entire process of regulation of the immune response in pregnancy is still under discussion. The present review focuses on facts showing that the progesterone is a kind of 'regulator of regulators' in the decidua, thus creating the most favourable conditions for the development of the semi-allogeneic fetus in successful pregnancy.

Drug-induced expression of nonsteroidal anti-inflammatory drug-activated gene/macrophage inhibitory cytokine-1/prostate-derived factor, a putative tumor suppressor, inhibits tumor growth.

A common in vitro response for many chemopreventive and antitumor agents, including some cyclooxygenase inhibitors, is the increased expression of nonsteroidal anti-inflammatory drug-activated gene (NAG)-1/macrophage inhibitory cytokine (MIC)-1/prostate-derived factor (PDF). The experimental anticancer drug 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F203) was a potent inducer of NAG-1 expression, and in MCF-7 cells, it inhibited cell growth and induced apoptosis. NAG-1 small interfering RNA blocked NAG-1 expression and 5F203-induced apoptosis in MCF-7 cells, indicating that NAG-1 may mediate the apoptosis and anticancer activity. One mechanism by which 5F203 increases NAG-1 expression is by increasing the stability of NAG-1 mRNA, dependent of de novo protein synthesis. Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was increased by 5F203, and inhibition of ERK1/2 phosphorylation abolished the induction of NAG-1 protein expression and increased the stability of NAG-1 mRNA. Thus, 5F203 regulates NAG-1 expression by a unique mechanism compared with other drugs. A mouse orthotopic mammary tumor model was used to determine whether 5F203 increased NAG-1 expression in vivo and suppressed tumor growth. Treatment of the mice with Phortress, the prodrug of 5F203, increased the in vivo expression of NAG-1 as measured by real-time reverse transcription-polymerase chain reaction from RNA obtained by needle biopsy, and the expression correlated with a reduction of tumor volume. These results confirm that NAG-1 suppresses tumor growth, and its in vivo expression can be controlled by treating mice with anticancer drugs, such as Phortress. Drugs that target NAG-1 could lead to a unique strategy for the development of chemotherapeutic and chemopreventive agents.

Regeneration and tolerance factor: a novel mediator of glioblastoma-associated immunosuppression.

Regeneration and tolerance factor (RTF) was originally identified in placenta where it is thought to be essential for fetal allograft survival. Here we report that RTF mRNA and protein are also expressed in human glioma cells in vitro and in vivo. Suppression of RTF expression by RNA interference promotes the lysis of glioma cells by natural killer (NK) and T cells in vitro. Moreover, RTF-depleted glioma cells are less tumorigenic than control cells in nude mice in vivo. Depletion of NK cells in these animals abolished this effect. RTF is thus a novel aberrantly expressed molecule which confers immune privilege to human malignant gliomas.

Glucocorticoid involvement in suppression of NK activity following surgery in rats.

We studied plasma factors mediating suppression of NK activity (NKA) following surgery. Plasma from operated rats suppressed NKA of splenocytes, leukocytes, and purified natural killer (NK) cells, and charcoal stripping nullified suppression. The glucocorticoid antagonist mifepristone prevented suppression, whereas blockers of reactive oxygen metabolites, opioids, catecholamines, prostaglandin-E2, and histamine did not. NKA dropped as corticosterone levels peaked postoperatively, and administration of relevant doses of corticosterone suppressed NKA. Inhibition of glucocorticoid synthesis prevented plasma from suppressing NKA but merely attenuated NKA suppression in operated rats. Thus, postoperative concentrations of corticosterone can directly suppress NKA but additional factors probably act in vivo.

Soluble fibrinogen-like protein 2/fibroleukin exhibits immunosuppressive properties: suppressing T cell proliferation and inhibiting maturation of bone marrow-derived dendritic cells.

Fibrinogen-like protein 2 (fgl2)/fibroleukin is a member of the fibrinogen-related protein superfamily. In addition to its established role in triggering thrombosis, it is known to be secreted by T cells. The soluble fgl2 ((s)fgl2) protein generated in a baculovirus expression system bound to both T cells and bone marrow-derived dendritic cells (DC) in a specific manner. (s)fgl2 exhibited immunomodulatory properties capable of inhibiting T cell proliferation stimulated by alloantigens, anti-CD3/anti-CD28 mAbs, and Con A in a dose-dependent manner; however, it had no inhibitory effects on CTL activity. The time- and dose-dependent inhibitory effect of (s)fgl2 on alloreactive T cell proliferation could be neutralized by a mAb against mouse fgl2. Polarization toward a Th2 cytokine profile with decreased production of IL-2 and IFN-gamma and increased production of IL-4 and IL-10 was observed in (s)fgl2-treated allogeneic cultures. Exposure of immature DC to (s)fgl2 abrogated the expression of CD80(high) and MHC class II(high) molecules and markedly inhibited NF-kappaB nuclear translocation, thus inhibiting their maturation. (s)Fgl2-treated DC had an impaired ability to stimulate allogeneic T cell proliferation. Maximal inhibition of proliferation was observed when allogeneic T cells were cultured with (s)fgl2-treated DC and (s)fgl2 protein was added in the culture. These data provide the first evidence to demonstrate that (s)fgl2 exerts immunosuppressive effects on T cell proliferation and DC maturation.

Effect of suppressive DNA on CpG-induced immune activation.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5'-->3' direction, and that a 5' motif can block recognition of immediately 3' sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs.

B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis.

Human endothelial cells (ECs) provide costimulatory signals sufficient to activate resting memory T cells to produce IL-2 and IFN-gamma, at least in part through CD58-CD2 interactions. Recently, the B7-like molecule, B7-H1 (PD-L1), was described and shown to regulate T cell activation; however, there are conflicting reports on whether it stimulates or inhibits T cell cytokine synthesis. B7-H1 is not expressed constitutively by ECs; however, it is rapidly induced by IFN-gamma, and synergistically by IFN-gamma and TNF. In inflamed skin, B7-H1 is expressed by a subset of microvessels, and by keratinocytes, but is barely detectable in normal skin. Blocking the interaction of EC-expressed B7-H1 with its T cell ligand, programmed death-1 (PD-1), using a PD-1-Fc fusion protein, or by blocking B7-H1 expression with morpholino antisense oligonucleotides, augments expression of IL-2 and IFN-gamma, implicating B7-H1 as a negative regulator of cytokine synthesis. However, signaling through PD-1 does not affect induction of the activation markers CD25 or CD69 on T cells, suggesting that its effects are specific to cytokine synthesis. The suppressive effects of B7-H1 on cytokine expression are proportional to the strength of the primary stimulus, allowing for B7-H1 to determine the level of T cell activation in response to ECs. Our results demonstrate that B7-H1 negatively regulates cytokine synthesis in T cells activated by ECs.

Characterization of the phenotypic and lymphokine profile associated with strong CD8+ anti-HIV-1 suppressor activity (CASA).

A panel of 22 CD8+ T cell lines, with a broad range of CD8+ anti-HIV-1 suppressor activity (CASA) were generated from a single patient with HIV-1 infection. CD8+ T cell lines with either strong or weak CASA were examined and compared for cell surface and intracellular markers, constitutive chemokine and lymphokine mRNA levels and inducible lymphokine expression. Strong CASA significantly correlated with CD8+ T cell lines that highly coexpressed the molecule CD28+ (r=0.52, P=0.01) and Ki67+ (r=0.88, P=0.02), with strong CASA CD8+ T cell lines demonstrating significantly higher (P < 0.05) expression of CD8+CD28+ and CD8+Ki67+ compared to those with weak activity. No such correlations or findings were observed for the markers CD38, HLA-DR, CD57 or perforin. The Th1 cytokines were expressed at greater levels than the Th2 cytokines, with strong CASA significantly associated with an increased inducible level of IL-2 production (P=0.05). Constitutive RANTES, IP-10 and I-309 mRNA expression were significantly (P < 0.05) elevated in CD8+ T cell lines exhibiting strong CASA compared to those with weak CASA. There was no significant difference in the mRNA expression of the lymphokines IL-2, 4, 5, 8, 9, 10, 14, 15, or chemokines MIP-1alpha, MIP-1beta, MCP-1, and Ltn. Strong CASA was therefore associated with rapidly replicating CD8+ T cells of the phenotype CD8+CD28+Ki67+ that expressed greater levels of IL-2 and the ligands RANTES and I-309.

Expression of a novel protein by regenerating hepatocytes and peripheral blood lymphocytes.

Regeneration and tolerance factor (RTF) is a protein with immunosuppressive activity and is normally present in the thymus and placenta. RTF was measured in the livers of patients with regenerating nodules due to alcoholic cirrhosis and hepatitis C. RTF was expressed in the regenerating nodules of 26 patients with alcoholic cirrhosis. All patients with chronic hepatitis C without cirrhosis failed to express RTF. Flow cytometry revealed upregulation of RTF on the lymphocytes from alcoholic cirrhosis and downregulation in hepatitis C disease.

Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis.

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.

Tumour-induced suppression of immune response and its correction.

Immunosuppressive features of tumour cells are a major obstacle for immunotherapy of cancer. We recently noted that RENCA cells effectively interfere with the in vivo activation of RENCA-specific T cells. To unravel the underlying mechanism, we evaluated the influence of RENCA cells on a mixed-lymphocyte/ tumour reaction as well as an allogeneic mixed-lymphocyte reaction. We observed that RENCA cells were not directly immunosuppressive. Instead, they initiated deviation of an immune response in at least two independent directions: (i) expansion of a population of NK1.1+/CD3+ cells, which was accompanied by elimination of mainly CD4+ lymphocytes, and (ii) production of a leukocyte-derived inhibitory factor. Expression of the costimulatory molecule B7.1 by RENCA cells prevented induction of anergy, while expression of MHC class II molecules prevented expansion of NK1.1+ cells, which was accompanied by a significant decrease in cell death. Hence, an unimpaired response was observed only when RENCA cells expressed B7.1 plus MHC class II molecules. Thus, even if a tumour itself is not immunosuppressive, it can induce a strong deviation of the immune response. It is concluded that the first contact between elements of the immune system and the tumour cell can confer a severe bias on immunoregulatory circuits.

Multiple mechanisms of immune evasion can coexist in melanoma tumor cell lines derived from the same patient.

Progressive tumor growth may be associated with suppression of the immune response. Many different mechanisms may contribute to immune evasion. We investigated some of these mechanisms in melanoma cells lines generated from two patients. These cell lines show a complex pattern of altered HLA expression; however, the resulting phenotype did not satisfactorily explain the simultaneous evasion of T and NK cell cytotoxicity. Two additional alterations have now been detected in these melanoma cell lines: (1) resistance to FAS-induced apoptosis caused by defective FAS gene expression, and (2) constitutive expression of immunosuppressive cytokines. Our results show that several of the major mechanisms for immune evasion may coexist in a single tumor. This suggests that tumor progression may give rise to an extremely resistant phenotype, which may be an impediment to some immunotherapeutic strategies. We hypothesize that the simultaneous presence of several mechanisms involved in tumor immune evasion must be the result of progressive selection of characteristics that are advantageous for tumor survival in a competent host. Our findings do not support the possibility that FASL expression is a common mechanism of evasion of immune response in melanoma cells.

Expression of regeneration and tolerance factor correlates directly with human immunodeficiency virus infection and inversely with hepatitis C virus infection.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3(+) T cells of HIV-seropositive (HIV(+)) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV(+) HCV-seropositive (HCV(+)), HIV- and HCV-seropositive (HIV(+) HCV(+)), and HIV- and HCV-seronegative (HIV(-) HCV(-)) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV(+) individuals is upregulated and that its expression on T cells from HCV(+) individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3(+) T cells was seen in both HIV(+) and HIV(+) HCV(+) individuals compared to HIV(-) HCV(-) individuals. HCV(+) individuals had lower levels of RTF expression than HIV(-) HCV(-) individuals (P < 0.005 for CD4(+); P < 0.0005 for CD8(+)). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV(+) > HIV(+) HCV(+) > HIV(-) HCV(-) > HCV(+). The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.

Generation of CD8+ T cell-generated suppressor factor and beta-chemokines by targeted iliac lymph node immunization in rhesus monkeys challenged with SHIV-89.6P by the rectal route.

The targeted lymph node (TLN) immunization strategy was investigated in macaques, in order to determine the efficacy in generating secretory, systemic, and cellular immune responses, CD8+ T cell-generated suppressor factors, and beta-chemokines. TLN immunization of the rectal and genital mucosa-associated iliac lymph nodes (TILNs) was compared with axillary TLN immunization (TAxLN) using HIV-1 MN/LAI gp140env and SIV p27gag in alum. Significantly higher immune responses, as well as CD8+ T cell-generated anti-SIV factors and the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta, were elicited by iliac as compared with axillary TLN immunization. The immune responses induced by TLN immunization were examined for their capacity to prevent rectal mucosal infection by the pathogenic dual-tropic SHIV-89.6P. Despite significant secretory, serum, cellular, and beta-chemokine responses, the macaques were infected by SHIV-89.6P. Whether the lack of protection was associated with the antigenic unrelatedness of SHIV-89.6P to the immunizing HIV-1 MN/LAI gp140 or to the virus utilizing CXCR4 to a much greater extent than CCR5, remains to be determined.