Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.

Polyoma middle T antigen (mT) was expressed in rat F-111 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promoter. Graded phenotypic responses to levels of mT induction by the hormone were seen, with morphological transformation, focus formation, and anchorage-independent growth requiring increasing levels of mT expression. The ability of different clones to form tumors reflected their maximum level of induction of mT-associated kinase and their ability to grow in soft agar. Expression of transformation parameters and tumorigenicity correlates with the level of mT phosphorylated by pp60c-src in immune complexes and not with the total amount of mT determined by metabolic labeling. We suggest that cellular factors regulate mT activity by forming a kinase-active fraction of mT molecules that controls the transformed state.     

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector.

The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week-old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1.     

Accelerated mammary tumor development in mutant polyomavirus middle T transgenic mice expressing elevated levels of either the Shc or Grb2 adapter protein

The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling.     

Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen.

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.     

Expression of matrix metalloproteinase 9 in pancreatic ductal carcinoma is associated with tumor metastasis formation

The objective of the current study was to assess the expression of matrix metalloproteinase 9 (MMP-9) in pancreatic ductal carcinoma and to examine its correlation with chosen clinico-anatomical parameters. The study group consisted of 36 patients with pancreatic ductal carcinoma. Tumors were stained using immunohistochemical method (NCL -MMP-9, Novocastra). No correlation was found between tumor MMP-9 expression and age, gender or grade of histological malignancy. However, statistical analysis revealed a relationship between tumor MMP-9 expression and histological type (adenocarcinoma mucinosum) of pancreatic carcinoma. The expression was strongly correlated with lymph node involvement and occurrence of distant metastases (p<0.00001). The results indicate a correlation between the expression of MMP-9 in pancreatic ductal carcinoma and worse prognosis (shown by lymph node involvement and distant metastases).     

The amount and activity of active matrix metalloproteinase 13 is suppressed by estradiol and progesterone in human pelvic floor fibroblasts

As a key degrader of fibrillar collagens, matrix metalloproteinase 13 (MMP13), may contribute to the progression of pelvic organ prolapse. Here we aimed to define the regulation of MMP13 by estradiol and progesterone in the vaginal supportive tissues. Fibroblasts cultured from the arcus tendineous fasciae pelvis of three pre- and three postmenopausal women with prolapse were treated with 17-beta-estradiol (E2), progesterone (P4), E2 + P4, or E2 + ICI 182,780 (ICI). Collagenase inhibitor I (CI) and MG-132 were employed to investigate the mechanism of MMP13 degradation into inactive fragments (fragmentation) by hormones. The regulation of MMP13 in vivo was assessed by comparing tissues of ovariectomized (ovx) vs. sham-operated rats. Expression of MMP13 (proenzyme and active and fragment forms) was quantitated by Western immunoblotting, and MMP13 enzymatic activity was measured using a substrate degradation assay. The amount of cellular active MMP13 and MMP13 proteolytic activity decreased in the presence of hormones. The decrease was paralleled by increased proenzyme and fragment forms. MG-132, not CI, suppressed cellular MMP13 fragmentation. Active MMP13 increased in rats following ovx and was suppressed by E2 + P4 supplementation. Active MMP13 is suppressed in vivo and in vitro by estradiol and progesterone, suggesting a protective effect against vaginal supportive tissue deterioration.     

12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function.

The 58,000-Mr form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes (B.S. Schaffhausen and T.L. Benjamin, J. Virol. 40:184-196, 1981). Here we report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60c-src. These results are discussed in terms of a possible role for protein kinase C in activating mT function(s), including the formation of stable complexes with pp60c-src.     

SV40 T antigen induced chromosomal changes reflect a process that is both clastogenic and aneuploidogenic and is ongoing throughout neoplastic progression of human fibroblasts.

In human fibroblasts, the expression of SV40 large T antigen is known to cause a variety of chromosomal aberrations and especially dicentric chromosomes. In some cases, the later aberrations have been reported to be reversible telomeric associations. We report here aberration and chromosome number studies of twenty-nine T antigen positive lineages, studied from their initiation by transfection of T antigen sequences into human diploid fibroblasts, until crisis or immortalization occurred or, in some cases until the lines became tumorigenic in nude mice. The data show that T antigen consistently produced chromosomal instability of both number and structure by an active process that began before transformation indicators were positive and continued throughout neoplastic progression. The most frequently observed aberrations were dicentric chromosomes, which were shown to be true dicentrics by examination by in situ hybridization with telomeric sequences. These data are consistent with the hypothesis that T antigen causes human fibroblasts to become neoplastically transformed by successive rounds of chromosomal mutation and lineage evolution.     

Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.     

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O(2)) than under 20% O(2) and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus, we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our (13)C NMR spectroscopic measurements indicate that (13)C-lactate is converted to (13)C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.     

TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8⁺ T cells in both chronic infection and tumor. However, the nature of TIM-3⁺CD4⁺ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4⁺ and CD8⁺ TILs from human lung cancer tissues but negligibly expressed on T cells from patients'' peripheral blood. Frequencies of IFN-γ⁺ cells were reduced in TIM-3⁺CD8⁺ TILs compared to TIM-3⁻CD8⁺ TILs. However, the level of TIM-3 expression on CD8⁺ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3⁺CD4⁺ TILs expressed FOXP3 and about 60% of FOXP3⁺ TILs were TIM-3⁺. Importantly, TIM-3 expression on CD4⁺ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.     

Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma

AIMS: To evaluate the expression of matrix metalloproteinase-19 (MMP-19) in oropharyngeal squamous cell carcinoma along with its association with structural features of invasiveness. To investigate whether MMP-19 expression correlates with lymphatic or systemic metastasis and prognosis in patients who have received definitive radiotherapy. METHODS AND RESULTS: The histological evaluation of the invasive front was based on Bryne's malignancy grading system. We correlated the immunohistochemical expression pattern with morphological parameters which characterize tumor invasiveness such as keratinization, nuclear polymorphism, invasion pattern, and the host inflammatory response. Local immunoreactivity for MMP-19 was positively correlated with tumor invasiveness as reflected in its structural characteristics and the degree of nuclear polymorphism, and negatively correlated with the inflammatory response of the host. No correlation existed between MMP-19 expression and clinicopathological features (TNM stage, grade of differentiation) or a patient's outcome and prognosis. CONCLUSIONS: This latter finding probably reflects the unique change for MMPs from high immunoreactivity within healthy tissue areas and non-invasive tumor parts, through absence in the least invasive neoplastic regions, to strong re-expression at a highly invasive front of the same tumor. Our findings indicate that MMP-19 can be used as a marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.     

Enhanced synthesis of factor B induced by tumor necrosis factor in human skin fibroblasts is decreased by IL-4

IL-4 is a T cell-derived lymphokine that has multiple biologic functions, affecting B cells, T cells, mast cells, monocytes, macrophages, and hematopoietic progenitor cells. We report that IL-4 also affects human skin fibroblasts. These effects were primarily in modulating the effects of other mediators of inflammation on these cells, with IL-4 producing little or no effects by itself. Synthesis of C proteins in human skin fibroblasts is modulated by TNF and other mediators of inflammation. TNF increased synthesis of factor B by 18.6-fold; IL-4 reduced the TNF effect on factor B synthesis by 83%. This effect started with concentrations of IL-4 as low as 0.1 ng/ml. The effect of IL-4 on the TNF-induced increase in synthesis of factor B occurred after 2 h incubation, the time when the effects of TNF alone were first observed. IL-4 also abrogated the effects of IL-1 and LPS on factor B synthesis, but did not affect the increase in synthesis of factor B induced by IFN-gamma. In contrast to the pattern for effect of IL-4 on factor B synthesis, the TNF-induced rate of synthesis of C3 was augmented by IL-4. The specificity of the IL-4 effect was also apparent when comparing the effect of IL-4 on the TNF-induced synthesis of factor B to the effects on total protein synthesis (increased 1.5-fold) and the TNF-induced increases in synthesis in C1r, C1s, C1 inhibitor, C2, and factor H (no changes). The IL-4-induced decrease in synthesis of factor B was mediated at a pretranslational level and was dependent on synthesis of a new protein. This interaction between IL-4 and mediators of inflammation can potentially modulate the inflammatory response in the setting of activation of Th cells.     

Selective recruitment of regulatory T cell through CCR6-CCL20 in hepatocellular carcinoma fosters tumor progression and predicts poor prognosis

BackgroundRegulatory T cells (Tregs) are highly prevalent in tumor tissue and can suppress effective anti-tumor immune responses. However, the source of the increased tumor-infiltrating Tregs and their contribution to cancer progression remain poorly understood.ConclusionsThe CCL20-CCR6 axis mediates the migration of circulating Tregs into tumor microenvironment, which in turn results in tumor progression and poor prognosis in HCC patients. Thus, blocking CCL20-CCR6 axis-mediated Treg migration may be a novel therapeutic target for HCC.     

Active antitumor immunotherapy, with or without B7-mediated costimulation, increases tumor progression in an immunogenic murine T cell lymphoma model

 BW-Sp3 is a BW-5147-derived T cell lymphoma with limited immunogenicity since, despite regression of the majority of subcutaneous tumors, an important fraction of the animals will die from metastases. In the present study, the BW-Sp3 cells were transfected with genes encoding B7-1 or B7-2, known to be involved in the induction of T cell responses. The resulting transfectants exhibited a reduced tumorigenicity and did not cause mortality in the syngeneic recipients. Furthermore, immunization with the B7-1 or B7-2 transfectants resulted in an increased generation of cytotoxic T lymphocytes (CTL) that lysed both the transfectants and the wild-type BW-Sp3 cells. Since the B7 transfectants were completely rejected in syngeneic recipients and induced potent CTL recognizing the wild-type BW-Sp3 cells, these engineered cells were considered as candidates for immunotherapy. Vaccinations with the B7-1 or B7-2 transfectants could completely protect the animals from metastatic disease when subsequently challenged with wild-type BW-Sp3 cells. Furthermore, immunization with the B7 transfectants could prolong the survival time of mice that had been challenged intravenously with BW-Sp3 cells. Surprisingly, however, when these transfectants, as well as the wild-type BW-Sp3 cells, were used for vaccination of tumor-bearing animals, the presence of the subcutaneous BW-Sp3 tumors clearly interfered with the outcome of immunotherapy, resulting in increased malignancy, as reflected by a higher incidence of progressing tumors and a reduced survival rate. Possible implications for immunotherapy in humans are discussed. Received: 5 August 1997 / Accepted: 15 August 1997     

Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion.     

Suppression of tumor growth and invasion in 9,10 dimethyl benz(a) anthracene induced mammary carcinoma by the plant bioflavonoid quercetin

Administration of quercetin, a common polyphenolic component of many vascular and edible plants including vegetables, fruits and tea significantly reduced the tumor volume in rats induced for mammary carcinoma using dimethyl benz (a) anthracene (DMBA). Dose response was assessed, by treating the animals with different doses (15-45 mg/kgbw) of quercetin and 25 mg/kgbw was taken as effective dose. Quercetin was administered as an intra tumoral injection once a week for 4 weeks. Serum levels of carcino embryonic antigen (CEA), a potent marker for tumor growth and invasion was significantly decreased on quercetin treatment. Quercetin caused a significant decrease in the activities of acid phosphatase and Cathepsin D in serum of experimental animals. Activities of lysosomal enzymes- (beta-D galactosidase, beta-D glucuronidase, beta-D glucosidase and sialidase), in serum and tissue were significantly altered in DMBA animals compared to control animals. However, quercetin treatment caused no significant change in lysosomal enzyme activities in tissues, whereas the activities were significantly lowered in serum. Partial purification of tissue type plasminogen activator (t-PA) from the tumor and kidney showed increased activity in the DMBA induced animals. Serum urokinase, -like plasminogen activator (u-PA) was also increased in animals with tumor, indicating tumor invasion. Administration of quercetin caused a significant decrease of both t-PA and u-PA. In conclusion, the present study suggests the possible role of quercetin in primary and invasive mammary tumor treatment. The above observations in vivo warrant further studies, due to the easy availability, common occurrence and low toxicity of this dietary bioflavonoid.