Tumor-Infiltrating Lymphocytes Express Vascular Endothelial Growth Factor in Renal Cell Carcinomas.

BACKGROUND: Known influence of the immune system on metastases of renal cell cancer (RCC) has led to the development of several therapeutic approaches for further stimulation of the host immune system by cytokines and the retransfusion of tumor-infiltrating lymphocytes (TIL). Based on observations in human prostate adenocarcinomas and transitional cell carcinomas, we have investigated the presence of TIL in different stages of RCC in correlation to vascular endothelial growth factor (VEGF) expression as a parameter for tumor progression and adverse survival in RCC patients. METHODS: Samples from surgically obtained RCC (n = 28) and benign renal parenchyma (n =10) were snap-frozen and processed by double-immunofluorescence staining with CD4/CD8 and VEGF antibodies. RESULTS: In 20 of 28 RCCs a coexpression of TIL-specific markers CD4 and/or CD8 and VEGF was demonstrated. Control tissues were VEGF-negative and showed only negligible infiltration by CD4- or CD8-positive lymphocytes. CONCLUSION: The results indicate that at least 71% of TIL produce VEGF and may promote tumor progression rather than represent an abortive antitumor response of the host immune system. Copyright 2000 S. Karger GmbH, Freiburg

DPC4/SMAD4 mediated tumor suppression of colon carcinoma cells is associated with reduced urokinase expression.

We recently identified DPC4/Smad4 as a candidate tumor suppressor gene mutated or lost in one half of pancreatic carcinomas and in a subset of colon and biliary tract carcinomas. DPC4 plays a key role in signal transduction of the TGF-beta superfamily of molecules and inactivation of TGF-beta mediated growth inhibition is supposed to be the driving force for DPC4 inactivation in human tumors. However, DPC4 mediated tumor suppression by reconstitution of defective cells has not yet been reported. Here we show suppression of tumorigenicity in nude mice by stable reexpression of DPC4 in SW480 colon carcinoma cells. In vitro growth of DPC4-transfected cells was not affected and resistance towards TGF-beta mediated growth inhibition was retained. Instead, cells exhibited morphological alterations and adhesion and spreading were accelerated. These phenotypic changes were associated with reduced expression levels of the endogenous urokinase-type plasminogen activator (uPA) and plasminogen-activator-inhibitor-1 (PAI-1) genes, the products of which are implicated in the control of cell adhesion and invasion. In patients, high expression levels of uPA and PAI-1 correlate with poor prognosis. Thus, reduced expression of uPA and PAI-1 is consistent with suppression of tumorigenicity in DPC4 reconstituted cells. These results demonstrate DPC4's tumor suppressive function and suggest a potential role for DPC4 as a modulator of cell adhesion and invasion.

Neural cell surface differentiation antigen gp130(RB13-6) induces fibroblasts and glioma cells to express astroglial proteins and invasive properties.

Transient expression of the differentiation and tumor cell surface antigen gp130(RB13-6) characterizes a subset of rat glial progenitor cells susceptible to ethylnitrosourea-induced neurooncogenesis. gp130(RB13-6) is as a member of an emerging protein family of ecto-phosphodiesterases/nucleotide pyrophosphatases that includes PC-1 and the tumor cell motility factor autotaxin. We have investigated the potential role of gp130(RB13-6) in glial differentiation by transfection of three cell lines of different origin that do not express endogenous gp130(RB13-6) (NIH-3T3 mouse fibroblasts; C6 and BT7Ca rat glioma cells) with the cDNA encoding gp130(RB13-6). The effect of gp130(RB13-6) expression was analyzed in terms of overall cell morphology, the expression of glial cell-specific marker proteins, and invasiveness. Transfectant sublines, consisting of 100% gp130(RB13-6)-positive cells, exhibited an altered, bipolar morphology. Fascicular aggregates of fibroblastoid cells subsequently developed into mesh-like patterns. Contrary to the parental NIH-3T3 and BT7Ca cells, the transfectant cells invaded into collagen type I. As shown by immunofluorescence staining of the transfectant sublines as well as of primary cultures composed of gp130(RB13-6)-positive and -negative cells, expression of gp130(RB13-6) induced coexpression of proteins typical for glial cells and their precursors, i.e., glial fibrillary acidic protein, the low affinity nerve growth factor receptor, and the neural proteins Thy-1, Ran-2, and S-100. In accordance with its expression in the immature rat nervous system, gp130(RB13-6) may thus have a significant role in the glial differentiation program and its subversion in neurooncogenesis.

gp130RB13-6-positive neural progenitor cells are susceptible to the oncogenic effect of ethylnitrosourea in pre-natal rat brain.

Proliferation-competent rat brain precursor cells of glial lineages are thought to preferentially undergo malignant transformation after transplacental exposure to ethylnitrosourea (EtNU). We recently have reported that monoclonal antibody (mAb) RB13-6 recognizes a developmentally regulated 130 kDa cell surface glycoprotein (gp130RB13-6) transiently expressed by a small subpopulation of glial progenitor cells in pre-natal rat brain. The expression of gp130RB13-6 has now been analysed immunocytochemically in malignant gliomas induced on day 15, 18 or 21 of gestation and in long-term cultures of fetal brain cells (FBC) isolated after in vivo-exposure to EtNU on day 18 of gestation. Malignant gliomas induced at different gestational stages contained varying proportions of gp130RB13-6-positive cells, whereas a subpopulation of proliferative neural progenitor cells exhibiting sustained gp130RB13-6 expression persisted in long-term FBC cultures after 3 months. This subpopulation, which was not selected for in control cultures of FBC derived from buffer-treated rats, gave rise to malignant cell lines after a period of time similar to the latency period required for glioma development in vivo. These data suggest that gp130RB13-6-positive cells of the immature rat nervous system may represent a subset of neural progenitor cells particularly susceptible to the oncogenic effect of EtNU.

Malignant transformation by cyclin E and Ha-Ras correlates with lower sensitivity towards induction of cell death but requires functional Myc and CDK4.

We demonstrate in this paper that the G1 phase specific cell cycle regulator cyclin E is able to provoke focus formation when cotransfected with activated Ha-ras into primary rat embryo fibroblasts (REFs). Cyclin E/Ha-ras transformed cells are highly tumorigenic in synergeneic rats, are able to form colonies in soft agar and show protection towards apoptosis upon serum starvation or DNA damage compared to cells transformed by the combination of Myc, cyclin D1 or SV40 large T-antigen and Ha-ras. Lines that were established after cyclin E/Ha-ras or cyclin D1/Ha-ras transformation contain a large percentage of polyploid cells. This was not observed in cells transformed with other oncoproteins and Ha-ras pointing to an involvement of D- and E type cyclins in genomic instability. The cyclin dependent kinase inhibitors p21 and p27 but also p16 completely abrogate focus formation by cyclin E and Ha-ras suggesting that the oncogenic activity of cyclin E still requires functional G1 specific cyclin/CDK complexes. Moreover, inhibition of Myc function also blocks the oncogenic activity of cyclin E indicating a requirement of Myc for cyclin E function. The findings presented here demonstrate that cyclin E can act as an oncoprotein with a potential involvement in genomic instability and the prevention of cell death. Our data also present more evidence for a strict functional interdependency between G1 cyclin/CDK complexes and c-Myc.

In vitro differentiation of neural progenitor cells from prenatal rat brain: common cell surface glycoprotein on three glial cell subsets.

Glial progenitor cell differentiation and cell lineage relationships during brain development are complex hierarchical processes depending on genetic programming, cell-cell interactions, and microenvironmental factors. The identification of precursor cell-specific antigens provides a tool for the study of both normal development and deviations from lineage-specific differentiation associated with malignant transformation. Monoclonal antibody (mAb) RB13-6 recognizes a 130-kDa cell surface glycoprotein (gp130RB13-6) expressed by a subset of 9OAcGD3-positive glial precursor cells scattered in the rat neuroepithelium on prenatal day (PRD) 13. During prenatal development the fraction of gp130RB13-6-positive fetal brain cells (FBC) decreased from about 18% (PRD 14) to about 1.5% (PRD 22), coinciding with increasing fractions of more mature cell types, as indicated by the elevated expression of p24RB21-15, another cell surface determinant specified by mAb RB21-15 (Kindler-Röhrborn et al.; Differentiation 30:53-60, 1985) and other neural cell type-specific markers. Accordingly, gp130RB13-6 positive precursor cells were localized in the ventricular zones throughout brain development. Concomitant with their formation and in the adult rat brain, ependymal layers lining the ventricular surface, choroid plexus, and the leptomeninges were intensely labeled by anti-gp130RB13-6 mAb. As visualized by confocal laser scanning microscopy of FBC cultures from PRD 13, gp130RB13-6 was coexpressed with the RC1 antigen by progenitor cells morphologically resembling radial glia cells. In addition, a very small subpopulation of astrocytes coexpressing gp130RB13-6 and glial fibrillary acidic protein (GFAP; < 5%) occurred 3 days after seeding. Primary FBC cultures from PRD 18 contained an increased subset of astrocytes coexpressing gp130RB13-6 and GFAP (approximately 25% of all gp130RB13-6 expressing cells), apparently generated from gp130RB13-6-positive precursors. Corresponding to in vivo conditions, ciliated ependymal cells but also microglial cells/macrophages and leptomeningeal cells showed strong expression of gp130RB13-6 in culture. We thus present a new glycoprotein on the cell surfaces of a glial progenitor cell subset for further studies of cell lineage relationships between radial glia cells, astrocytes, and ependymal cells.

Influence of elevated expression of rat wild-type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth-arrest-specific gene gas3. The biological function of PMP22 is unknown, but recent progress in the analysis of rat Schwann cells expressing altered levels of PMP22 revealed that one role of PMP22 is as a negative growth modulator. We have investigated the influence of rat PMP22 (rPMP22) and a mutant of PMP22 (rPMP22(Tr)) resembling the murine trembler mutation on cell growth of retrovirus-vector-infected mouse NIH3T3 cells. Transduced cells carrying the two different sense constructs expressed rPMP22 and rPMP22(Tr )mRNAs and proteins. Elevated levels of rPMP22 and rPMP22(Tr )significantly reduced fibroblast growth as judged by proliferation assays. Despite a negative modulatory influence of rPMP22 and rPMP22(Tr )on cell proliferation, cell cycle analyses by flow cytometry did not reveal an influence of rPMP22 or rPMP22(Tr )on the synchronous progression of resting NIH3T3 cells from G0 into S phase. However, cell cycle analyses by flow cytometry of asynchronously dividing cultures demonstrated that the expression of rPMP22 and rPMP22(Tr )increased the fraction of cells in the G1 phase of the cell cycle. Furthermore, cell death analyses revealed that, in contrast to control cells and cells carrying the rPMP22(Tr )construct, a significantly increased fraction of NIH3T3 cells expressing rPMP22 exit the proliferation compartment showing hallmarks of programmed cell death. These results indicate that (i) rPMP22 and rPMP22(Tr )act as negative modulators of proliferation in murine fibroblasts probably through extension of the G1 phase of the cell cycle and (ii) rPMP22 but not rPMP22(Tr )promotes programmed death of these cells.

Characterization of rat NCA/CD9 cell surface antigen and its expression by normal and malignant neural cells.

As part of investigations on ethylnitrosourea (EtNU)-induced neuro-oncogenesis in the rat, we have produced monoclonal antibodies (Mabs) specific for neural cell surface antigens (NCAs) by immunization with cells of the clonal tumorigenic neural rat cell line BT4Ca. Mabs designated as anti-NCA (alpha NCA1, alpha NCA2, alpha NCA3, alpha NCA4, and alpha NCA5) recognize proteins of 25 kDa and 23 kDa, as shown by immunoprecipitation and Western blot. The predominant 25-kDa protein was purified from BT4Ca cells by immunoaffinity chromatography with immobilized Mab alpha NCA1 and identified by N-terminal sequencing as the rat homologue of the CD9 antigen. Identification of proline as N-terminal amino acid of the purified protein suggests post-translational modification of CD9 in the rat central nervous system. The NCA/CD9 protein was localized in distinct regions of fetal and adult rat brain by immunofluorescence staining of frozen sections. Flow cytometric analyses of isolated fetal rat brain cells (FBC) showed that the proportion and number of NCA/CD9-expressing cells increased during prenatal development. Immunoreactivity of approximately 40% of brain cells isolated 13 days post conception (p.c.) indicated that NCA/CD9 is expressed by neuronal precursors at this stage of development. In primary cultures of rat FBC isolated 18 days p.c., the NCA/CD9 antigen was expressed by all premature and mature astrocytes, oligodendrocytes, ependymal cells, and microglial cells, but not by E-N-CAM-expressing neuronal progenitor cells and neurons. Furthermore, eight out of ten EtNU-induced malignant neural rat cell lines as well as EtNU-induced tumors of the central and peripheral nervous system exhibited intermediate or strong immunoreactivity with Mab alpha NCA1. Expression of the NCA/CD9 protein is, therefore, characteristic of both normal glial precursor cells and their malignant counterparts in the rat.

Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth arrest-specific gene gas3. Besides a putative role of PMP22 in myelination, a regulatory function in cell growth has been suspected. Here we have investigated both the expression of PMP22 during cell cycle progression of cultured rat Schwann cells and the influence of altered levels of PMP22 on Schwann cell growth. When resting cells were stimulated to begin the cell cycle, the regulation of PMP22 mRNA resembled the growth arrest-specific pattern of gas3 expression observed previously in NIH3T3 fibroblasts. To prove a growth regulatory function of PMP22, we generated Schwann cell cultures by infection with retroviral PMP22 expression vectors that constitutively expressed PMP22 cDNA sequences, in either the sense or antisense orientation. Transduced cells carrying the sense construct overexpressed PMP22 mRNA and protein, whereas in cells infected with an antisense PMP22 expression vector PMP22 mRNA levels were reduced markedly. Altered levels of PMP22 significantly modulated Schwann cell proliferation, as judged by 5-bromo-2'-deoxy-uridine incorporation into replicated DNA. In asynchronously dividing cultures enhanced expression of PMP22 decreased DNA synthesis to 60% of the control level. Conversely, reduced levels of PMP22 mRNA led to enhanced DNA synthesis of approximately 150%. Further cell cycle analyses by flow cytometry revealed that overexpression of PMP22 delayed serum- and forskolin-stimulated entry of resting Schwann cells from G0/G1 into the S + G2/M phases by approximately 8 h, whereas underexpression of PMP22 mRNA slightly increased the proportion of cells that entered the S + G2/M phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface antigens of cell subpopulations in prenatal rat brain are expressed in a characteristic non-random pattern on their ethylnitrosourea-induced malignant counterparts.

Selective induction of neural tumors in the rat by single-dose exposure of the immature nervous system to ethylnitrosourea (EtNU) is a model for the study of cell lineage-, differentiation stage-, and carcinogen-dependent mechanisms in neuro-oncogenesis. Overall yields and relative frequencies of different types of neural tumors vary with the developmental window chosen for the EtNU-pulse. Precursor cells belonging to different neural lineages and targeted by the carcinogen at distinct developmental stages may thus bear a differential risk of malignant conversion. To specify subpopulations of neural precursors in fetal (prenatal day 18) BDIX-rat brain, four monoclonal antibodies (mAbs) recognizing cell surface differentiation antigens were used: mAb RB13-2 directed against O-acetylated gangliosides and binding to approximately 36% of fetal brain cells (FBC); mAb RB13-6 recognizing a 130 kDa glycoprotein (expressed by approximately 8% of FBC); and mAbs RB21-7 and RB21-15 which bind, respectively, to embryonal neural cell adhesion molecules (N-CAM) and a 24 kDa protein (expressed by approximately 55% and 12% of FBC). Antigen expression profiles were compared with those of 14 primary brain tumors and 16 malignant neural cell lines, all of which had been induced by EtNU on prenatal day 18 in vivo. Monoclonal antibodies RB13-2 and RB21-7 did not bind to any of the tumors or cell lines. In contrast, mAbs RB13-6 and RB21-15 both reacted with 14/14 tumors, and with 16/16 and 10/16 cell lines, respectively. Expression of the latter antigens might thus specify lineage-specific stages of FBC development/differentiation particularly susceptible to EtNU-induced malignant transformation. Two-color fluorescence analyses revealed three subsets of FBC binding mAb RB13-6 (RB13-2+/RB13-6+/RB21-15-; RB13-2-/RB13-6+/RB21-15-; and RB13-2-/RB13-6+/RB21-15+), representing successive stages of differentiation.

In vitro differentiation of E-N-CAM expressing rat neural precursor cells isolated by FACS during prenatal development.

Most fetal rat brain cells expressing the embryonal, highly sialylated form of the cell adhesion molecule N-CAM (E-N-CAM) are precursor cells, as judged from the absence of marker molecules specific for mature neural cell types. However, the detection of E-N-CAM+ cells in frozen sections does not provide information on the lineage-specific differentiation of these cells during development. To investigate their differentiation behaviour in vitro, E-N-CAM+ cells were isolated at different times of brain development by fluorescence-activated cell sorting (FACS), using a monoclonal antibody (Mab RB21-7) which specifically recognizes polysialic acid (PSA) residues on E-N-CAM. Double-immunofluorescence analyses showed that the majority of E-N-CAM+ cells isolated on prenatal days 15 to 18 differentiated into neurons while a small subset of Mab RB21-7 binding cells proved to be astrocytic precursors and/or bipotential. The proportion of E-N-CAM+ astrocytic precursors increased during later development (prenatal day 22) concomitantly with the onset of gliogenesis. While conversion of E-N-CAM to mature forms of N-CAM was never observed in neurons during cultivation, E-N-CAM+ cells of the astrocyte lineage switched to N-CAM soon after the onset of GFAP expression. A lineage-specific transition of E-N-CAM to mature N-CAM expression is, therefore, suggested for these astrocytic progenitor cells during rat brain development.