Expression and prognostic value of transcription factor PROX1 in colorectal cancer.

BACKGROUND: PROX1 is a specific target of the ß-catenin/TCF pathway in the intestinal epithelium. It acts as a regulator of progression from a benign to a highly dysplastic phenotype in colorectal tumours. However, the clinical significance of PROX1 expression is not known. METHODS: We studied the prognostic value of immunohistochemical expression of PROX1 in a series of 517 patients with colorectal cancer (CRC). RESULTS: The majority of the tumour samples expressed PROX1 (91%, 471 out of 517). High PROX1 expression was associated with a poor grade of tumour differentiation (P<0.0001). In the subgroup of patients with colon cancer, high PROX1 expression was associated with unfavourable colorectal cancer-specific survival (CCSS) as compared with low PROX1 expression (CCSS 47% vs 62%; P=0.045; RR 1.47). The association between high PROX1 and poor outcome was further strengthened in female colon cancer patients (CCSS 38% vs 63%; P=0.007; RR 2.02). Nonetheless, in multivariate survival analysis PROX1 expression was not retained as an independent prognostic factor. CONCLUSION: High PROX1 expression is associated with a poor grade of tumour differentiation, and, in colon cancer patients, also with less favourable patient outcome. Our results strengthen the previous preclinical observations that PROX1 has a role in tumour progression in CRC.

Antigen-binding T cells as helper cells. Separation of helper cells by immune rosette formation.

The spleen T cells from mice immunized 6 days earlier with either chicken gamma globulin (CGG) or with donkey erythrocytes (DRC) were rosetted with CGG-coated sheep erythrocytes or with DRC. The immune rosettes (RFC) (antigen-binding cells) were separated from the bulk of nonrosette-forming cells (non-RFC) by 1-g velocity sedimentation and the RFC and non-RFC tested for helper activity in cooperative antihapten responses in vitro. RFC or non-RFC were mixed with normal or hapten-primed spleen cells, challenged with the appropriate hapten-carrier conjugate and cultured for 4 days in Marbrook tissue cultures. The helping activity was quantitated from the numbers of antihapten antibody-producing cells generated per culture. The results show that specific helper cell activity could be selectively recovered in the immune rosette-forming cell population whereas the non-RFC population was depleted of help. These findings indicate that the helper T cells express specific antigen binding receptors.

Sequential responses of mouse spleen T cells in mixed lymphocyte culture-induced cytolysis.

T cells triggered to blast transformation and proliferation by histoincompatible cells have the capacity of reverting "back" to lymphocytes. These "secondary" lymphocytes and their progeny cells are able to respond repeatedly to the same allogeneic stimulus in vitro.

Sex hormone regulation of in vitro immune response. Estradiol enhances human B cell maturation via inhibition of suppressor T cells in pokeweed mitogen-stimulated cultures.

The effects of the main male and female sex hormones, testosterones and estradiol, in pokeweed mitogen (PWM)-stimulated cultures of human blood lymphocytes were studied. We found that the addition of physiological concentrations of estradiol (780-2,600 pmol/liter) to PWM cultures significantly increased the accumulation of immunoglobulin M-containing and -secreting cells detected by immunofluorescence and/or by the reversed protein-A plaque assay. The dose range of estradiol that induced enhanced B cell maturation did not affect the proliferative response. Estradiol displayed the same effect in vitro on lymphocytes from both men and women. Fractionation of lymphocyte subpopulations before culturing revealed that estradiol does not display a direct mitogenic or stimulatory effect of B cells. Instead, estradiol inhibits the suppressive activity of a radio-sensitive (1,000 rad) subset of T lymphocytes bearing Fc-receptors for immunoglobulin G. Nontoxic concentrations fo testosterone did not influence the in vitro B cell maturation. These observations provide a cellular basis for the differences in the immunoreactivities of males and females. The estradiol-induced inhibiton of suppressor T cells might be important for the pathogenesis of various autoimmune disorders.

Leukocyte surface origin of human alpha1-acid glycoprotein (orosomucoid).

Specific antibodies against human alpha1-acid glycoprotein reacted with human lymphocytes, granulocytes, and monocytes. The antigen on the leukocytes is an externally located integral membrane glycoprotein which is made by the cells and has an apparent mol wt of 52,000. It is released from cells in vitro to the culture medium. The mol wt of the soluble fragment is 41,000, which corresponds to that of alpha1-acid glycoprotein in serum and urine. Peptide mapping confirmed that the main part of the cellular membrane antigen consists of alpha1-acid glycoprotein with an additional, probably hydrophobic fragment. This finding may partially explain the increase in the serum levels of alpha1-acid glycoprotein observed in many disorders involving leukocyte proliferation. In addition, the known sequence homology of alpha1-acid glycoprotein with immunoglobulins can now be more easily understood by their origin in similar cell types.

Secondary in vitro responses of T lymphocytes to non-H-2 alloantigens self-H-2-restricted responses induced in heterologous serum are not dependent on primary-stimulating non-H-2 alloantigens.

The role of non-H-2 alloantigens, specifically Mls locus products, in secondary in vitro T-cell-mediated cytotoxicity has been studied. Splenic T lymphocytes, activated against Mls locus alloantigens in primary-mixed cultures and isolated by velocity sedimentation gradient separation techniques, were used as responding populations in secondary mixed leukocyte cultures (MLCs) and cell-mediated lympholysis (CML). Such T-cell clones could be shown to exhibit either "self"-H-2-restricted or anti-Mls locus-specific reactivity, with this dichotomy of reactivity depending only on the primary culture conditions. Mls locus-activated T lymphocytes generated in cultures supplemented with homologous serum exhibited specific memory responses in MLC, yet remained incapable of effecting target cell destruction against Mls locus antigens or against "self"-H-2-structures in CML. In contrast, activated T-cell clones generated in the presence of heterologous serum displayed H-2-restricted reactivity in both secondary MLC and CML. H-2-restricted MLC activation was controlled by products of the H-2 serologically defined regions. Although heterologous serum was a necessary (and sufficient) entity for development of H-2-restricted responses, evidence argues against the possibility that heterologous serum acts via modification of cell surface components.

Proliferation of B and T cells in mixed lymphocyte cultures.

Electrophoretically fractionated CBA/Ca spleen T cells alone respond to allogeneic cells in one-way MLC and to PHA. They do not respond to E. coli LPS. B cells alone do not respond to allogeneic cells nor to PHA, but do respond to LPS. When karyotypically distinguishable syngeneic mixtures of T and B lymphocytes are stimulated with allogeneic cells, at the most 5% of mitoses on 5-9th culture day are of B cell origin. This indicates that B cells are not substantially recruited to proliferate in the MLC.

Induction of specific immune unresponsiveness using purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. I. Demonstration of general validity as to species and histocompatibility barriers.

Normal immunocompetent T lymphocytes can be induced into specific proliferation if confronted with the relevant alloantigen in vitro. Such mixed leuko-cyteculture-activated T lymphoblasts carring idiotypic receptors on their surface can be purified using velocity sedimentation and serve as immunogen if administered in adjuvant to the autologous host. Autoblast immunization can be shown to lead to specific, long-lasting unresponsiveness against the relevant alloantigens, while leaving reactivity against third-party antigens intact. When tested as to general validity, it could be shown to function in all species analyzed (mouse, rat, and guinea pig) as well as across both major and minor histocompatibility barriers. No negative side effects have been noted so far. It would thus seem clear that autoblast immunization using the above described scheme may serve as a general tool in inducing long-lasting, specific unresponsiveness in any species and across any histocompatibility barrier.

Induction of specific immune unresponsiveness with purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. II. An analysis of the effects measured at the cellular and serological levels.

T lymphoblasts specific for foreign histocompatibility antigens and purified via mixed leukocyte culture (MLC) and 1 g velocity sedimentation procedures can be used as autoimmunogen to produce specific immunological unresponsiveness in adult animals. This unresponsiveness is positively correlated to the production of autoanti-idiotypic antibodies in the blast immunized animals and no evidence of coexisting alloimmunity was found. We consider this autoanti-idiotypic immunity to be the specific inducing agent of the immune tolerance. The blast immunization procedure will lead to selective reduction in T-cell reactivity against the relevant alloantigens as measured by MLC, cell-mediated lympholysis, or graft-versus-host assays. However, in individual animals, dichtomy in suppression between two T-cell assays could sometimes be observed indicating elimination of only a select group of idiotypic functionally distinct population of T cells in these blast-immunized animals. Attempts to abrogate already immune animals by the autoblast procedure were successful, in part suggesting the use of the present procedure when trying to induce in accelerated reversion of such immunity.

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype.

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v-src, and implicates a pivotal role for polyamines in cell transformation.

Characterization of surface glycoproteins of mouse lymphoid cells.

We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique. The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography). The major thymocyte surface proteins have molecular weights of 170,000 and 125,000. Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000. Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000. Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000. When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface. A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide. T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern. A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion.

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.

Thymus-dependent and thymus-independent lymphocyte separation: relation to exposed sialic acid on cell surface.

On preparative cell electrophoresis mouse lymph node lymphocytes separate into fast-moving (T, thymus-dependent) and slow-moving (B, thymus-independent) fractions. After treatment with neuraminidase all lymphocytes move as one very slow fraction, indicating that the difference in the mobility of the two kinds of cells is due to differences in the density of exposed sialic acid on their surfaces.

Ornithine decarboxylase- and ras-induced cell transformations: reversal by protein tyrosine kinase inhibitors and role of pp130CAS.

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src- and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.

Changes in cell surface glycoproteins and antigens during differentiation of the human myeloid leukemia cell lines ML-1, ML-2, and HL-60.

Two recently derived human myeloid leukemia cell lines, ML-1 and ML-2, were induced to differentiate by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or with retinoic acid for 5 to 12 days. They were then compared with similarly treated promyelocytic HL-60 cells. TPA-treated ML-1 and ML-2 cells became firmly surface adhesive with a fibroblastoid morphology, while TPA-treated HL-60 cells adhered as rounded macrophages. In contrast, retinoic acid induced only slight morphological changes in all three cell lines. The differentiation-related alterations of the surface membrane glycoproteins were followed by polyacrylamide gel electrophoresis after surface labeling by the periodate-NaB3H4 or galactose oxidase-NaB3H4 methods. The expression of surface membrane differentiation antigens was analyzed with a panel of monoclonal antibodies against myeloid, myelomonocytic, monocytic, and granulocytic determinants using FACS IV flow cytometry. The acquisition of surface adhesiveness by TPA-treated ML-1 and ML-2 cells coincided with the appearance of membrane surface proteins of varying molecular weights, ranging between 90,000 and 155,000, which were not labeled in untreated ML-1 and ML-2 cells. These findings and the results obtained by monoclonal antibody staining and FACS analysis indicate that treatment of the myeloid lines ML-1, ML-2, and HL-60 by TPA induced the expression of antigenic and membrane molecular features compatible with a monocytic-macrophage phenotype, while treatment by retinoic acid induced granulocytic differentiation. The ML-1 and ML-2 cells offer interesting models for studies on the membrane molecular events occurring when nonadherent, monocytic cells become surface adherent.

Metastasizing neuroblastomas in mice transgenic for simian virus 40 large T (SV40T) under the olfactory marker protein gene promoter.

The olfactory marker protein (OMP) is a M(r) 19,000 polypeptide originally considered a selective marker for differentiated olfactory receptor neurons. In an attempt to induce neoplastic proliferation in the olfactory cells, we made mice transgenic for the simian virus 40 large T-antigen gene linked to the OMP gene promoter. Four independent lines of transgenic mice were established. Despite a high expression of the transgene in the olfactory receptor neurons, no evidence of tumor growth was observed. Instead, starting from an age of 4 months, animals of all four lines presented with highly metastatic tumors originating in the adrenal medullas or sympathetic ganglia. The histological, ultrastructural, and immunohistochemical features of the tumors were identical to those of human infant neuroblastoma. Five independent neuroblastoma cell lines were established from tumors of different transgenic animals. All cell lines constitutively express the endogenous OMP gene. The transgene product, simian virus 40 large T-antigen, associates with the product of the anti-oncogene p53 in these cell lines. This transgene system not only offers a biologically faithful model for investigations on the pathogenesis of neuroblastoma, the most common solid neoplasia of infancy, it also raises intriguing questions about the role of the OMP gene for the differentiation of the sympathetic neurons.

DNA copy number losses in chromosome 14: an early change in gastrointestinal stromal tumors.

The DNA copy number changes were investigated in 13 malignant, 3 borderline, and 16 benign gastrointestinal stromal tumors (GISTs) by comparative genomic hybridization. A consistent finding was the loss of DNA copy numbers in chromosome 14q. This was detected in 75% of the benign tumors, in 62% of the malignant tumors, and in two out of the three borderline tumors with a minimal overlapping region located to 14q22. Losses of DNA copy numbers were more frequent than gains, with 2-10 changes in malignant GISTs and 1-3 changes in benign tumors. High-level DNA amplifications, detected only in malignant GISTs, were assigned to 3q26-q29 (40%), 5p (30%), and 8q22-24 (40%). Our results indicate that copy number losses in 14q are an early change during oncogenesis of GISTs and suggest the possibility that a new tumor suppressor gene at 14q22 might be involved in the regulation of differentiation or proliferation of such mesenchymal cells.

DNA sequence copy number changes in gastrointestinal stromal tumors: tumor progression and prognostic significance.

To identify genetic changes related to tumor progression and find out diagnostic and prognostic genetic markers in gastrointestinal stromal tumors (GISTs), 95 tumor samples (24 benign GISTs, 36 malignant primary GISTs, and 35 GIST-metastases) from 60 patients were studied using comparative genomic hybridization. DNA copy number changes were detected in all samples. Benign GISTs had a mean of 2.6 aberrations/ sample (losses:gains, 5:1) and significantly fewer DNA copy number changes and fewer gains than malignant primary and metastatic GISTs (P < 0.01). High-level amplifications were not seen in benign GISTs. Malignant primary GISTs had a mean of 7.5 aberrations/tumor (losses: gains, 1.6:1), whereas the mean number of aberrations/metastatic GIST was 9 (losses:gains, 1.8:1). Frequent changes observed in all GIST groups included losses in chromosome arms 1p (51%), 14q (74%), and 22q (53%). Gains and high-level amplifications at 8q and 17q were significantly more frequent in metastatic GISTs (57 and 43%) than in benign GISTs (8 and 0%; P < 0.001) and malignant primary GISTs (33 and 25%; P < 0.05). Gains and high-level amplifications at 20q were only seen in malignant primary and metastatic GISTs (P < 0.01), and gains at 5p were not detected in benign GISTs (P < 0.01). Losses in chromosome arm 9p were never seen in benign tumors (P < 0.001), and they were more frequent in metastatic GISTs than in malignant primary GISTs (63 and 36%; P < 0.05). Losses in 13q were less frequent in benign GISTs than in malignant primary (P < 0.05) and metastatic (P < 0.01) GISTs. Our results show that several DNA copy number changes are related to the behavior of GISTs and can be used as prognostic markers for tumor progression.

Human ornithine decarboxylase-overproducing NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice.

Overexpression of human ornithine decarboxylase (ODC) under the control of strong promoters induces morphological transformation of immortalized NIH3T3 and Rat-1 fibroblasts [M. Auvinen et al., Nature (Lond.), 360: 355-358, 1992]. We demonstrate here that ODC-overproducing NIH3T3 cells are tumorigenic in nude mice, giving rise to rapidly growing, large fibrosarcomas at the site of inoculation. The tumors are capable of invading host fat and muscle tissues and are vascularized abundantly. To disclose the molecular mechanism(s) driving the tumorigenic, invasive, and angiogenic phenotype of the tumors, the ODC-overproducing cell lines and tumor tissues were analyzed for the expression of various potential regulators and mediators of cell proliferation, matrix degradation, and angiogenesis. The tumorigenicity of ODC transformants was associated with elevated polyamine levels and down-regulated growth factor receptors. The invasiveness of the ODC-induced tumors could not be attributed to overexpression of various known extracellular matrix-degrading proteases or matrix metalloproteinases. The induction of the tumor neovascularization proved not to be elicited by vascular endothelial growth factor or basic fibroblast growth factor. Instead, the ODC-overexpressing cells appeared to secrete a novel angiogenic factor(s) that was able to promote migration of bovine capillary endothelial cells in collagen gels and increase the proliferation of human endothelial cells in vitro. In parallel, ODC-transformed cells displayed down-regulation of thrombospondin-1 and -2, the negative regulators of angiogenesis. Thus, the induction of the angiogenic phenotype of the ODC transformants is likely due both to increased expression and secretion of the new angiogenesis-stimulating factor(s) and decreased production and release of the antiangiogenic thrombospondins.

Expression of cyclin A in soft tissue sarcomas correlates with tumor aggressiveness.

Cyclins and cyclin-dependent kinases regulate the cell cycle. Cyclin A has a dual role in cell proliferation. It is essential in the S phase for DNA replication, and it is also involved in G2-M-phase transition, signifying actively dividing cells. The expression of cyclin A was determined by immunohistochemistry in paraffin sections of 126 soft tissue sarcomas. The median cyclin A score was 10.8% (range, 1-54%). Cyclin A expression correlated with the S-phase fraction, Ki-67 score, G2-M phase, and grade. It did not correlate with the size of the tumor. A high cyclin A score predicted a poor metastasis-free survival (P < 0.01) and a poor disease-specific overall survival (P = 0.01). We concluded that the expression of cyclin A is a powerful prognostic factor in soft tissue sarcoma. Moreover, the cyclin A score determines the fraction of tumor cells in the S phase and the G2 phase, which are the most sensitive cell cycle phases for current modalities of cancer treatment.