A regulatory element in the CD95 (APO-1/Fas) ligand promoter is essential for responsiveness to TCR-mediated activation.

Expression of the CD95 (APO-1/Fas) ligand (CD95L) in activated T cells is a major cause of T cell activation-induced apoptosis. To study the molecular mechanisms of transcriptional control of CD95L expression in T cells, we investigated the human CD95L promoter in Jurkat T cells. Deletion studies revealed that the CD95L proximal promoter sequence from -220 to the transcription start site is essential for T cell stimulation-induced expression of CD95L. In this study, we discovered a novel regulatory element located at -120 of the CD95L promoter which contains DNA binding sites for SP-1 and a yet unknown inducible factor. Mutation analysis demonstrated that binding of the inducible factor to the -120 region is crucial for the biological function of the CD95L promoter upon T cell stimulation. The DNA sequence at -120 also contains two DNA motifs homologous to the binding site for NF-AT. NF-AT does not directly bind to this element. However, cotransfection studies with an NF-AT expression vector showed that NF-AT may confer a strong inducible activity to the CD95L promoter at this regulatory region. Our data also show that the immunosuppressive agent cyclosporin A down-regulates CD95L transcription by inhibiting the function of this positive regulatory element.

Blockade of metastasis formation by CD44-receptor globulin.

CD44 standard as well as variant isoforms have been frequently reported to be involved in the process of metastasis formation. Whereas in the rat system, but also in some human tumours, the variant exon v6 is of importance in the lymphatic spread of carcinomas, in human malignant melanoma CD44s and, possibly, CD44v10 appear to facilitate local invasion and haematogenous spread. This has been tested in the B16F10 murine melanoma model by treating B16F10-bearing C57BL/6 mice either with a CD44s-/ CD44v10-specific antibody, or with receptor globulins (Rg) containing the extracellular part of CD44s or CD44v10 linked to the constant region of the immunoglobulin kappa light chain. Prior characterization of the CD44s and CD44v10 Rg had shown that both Rgs bound to components of the extracellular matrix, CD44s in particular to hyaluronic acid. Immunohistological screening of organ sections from adult C57BL/6 mice revealed additional evidence for both Rgs binding to elements of the extracellular matrix, particularly in bone marrow, intestine and lung. In the absence of any further treatment, the CD44s Rg reduced the number of lung colonies by 70%, while application of the CD44v10 Rg resulted in 60% reduction. CD44-specific antibodies were equally efficient with regard to B16F10 settlement in the lung. However, only the CD44 Rgs prevented spread and settlement of melanoma cells in distant organs. The finding confirms the involvement of both CD44s and CD44v10 in melanoma progression, and is suggestive for the use of Rgs as therapeutic reagents.

Efficient lysis of CD44v7/8-presenting target cells by genetically engineered cytotoxic T-lymphocytes--a model for immunogene therapy of cervical cancer.

Variant proteins of the CD44 surface glycoprotein family are expressed on many different human tumors and their lymph node metastases. An epitope encoded by sequences of variant exons CD44v7 and v8 and recognized by the monoclonal antibody VFF17 is frequently detected in cervical cancer, whereas the normal cervical epithelium lacks expression of this epitope. We have developed an immunotherapeutic approach for cervical cancer based on the expression of this CD44v7/8 epitope. The single chain antigen-binding fragment of VFF17 was fused to a signal transducing protein (zeta-chain) of the T-cell receptor complex (TCR) and was introduced into a retroviral gene transfer vector. Gene transfer was applied to the murine cytotoxic T-cell line cl96. All recombinant clones expressed the fusion protein on their cell surface. Functionality of the recombinant fusion protein was tested by subjection of several recombinant clones to in vitro cytotoxicity assays. CD44v7/8-expressing target cells were killed efficiently by reprogrammed cl96 in an MHC-independent fashion, whereas CD44v7/ 8-negative cells were not affected. These transfected T cell lines will now be tested in vivo using immune-deficient mice bearing CD44v7/8-expressing tumors.

Growth retardation of tumors by adoptive transfer of cytotoxic T lymphocytes reprogrammed by CD44v6-specific scFv:zeta-chimera.

Variants of the CD44 protein family containing sequences encoded by variant exon 6 (v6) are involved in the metastatic spread of rat and human tumors. The rat-specific antibody 1.1ASML, which recognizes a v6 epitope, interferes with metastatic dissemination of a rat pancreatic carcinoma. The single-chain antigen-binding fragment of this monoclonal antibody was fused to the zeta-chain of the T-cell receptor complex. The appropriate fusion gene was incorporated into a retroviral gene transfer vector. Murine cytotoxic T lymphocytes (CTLs) were infected, and cellular clones which express the single-chain zeta-chain fusion protein on their cell surface were selected. These CTLs are not MHC-restricted in their CD44v6 recognition and exhibit in vitro lytic activity toward cells expressing CD44 variants comprising exon v6. Tumor cell xenografts grown in athymic nude mice are suppressed in their growth upon infusion of the genetically manipulated CTLs. Our data indicate that the CD44v6 epitope is an effective target for immune tumor therapy and demonstrate the efficacy of genetically engineered CTLs in targeting tumors expressing such epitopes.

Hyaluronate-independent metastatic behavior of CD44 variant-expressing pancreatic carcinoma cells.

Several studies have demonstrated a correlation between the expression of CD44 variant isoforms and the ability of tumor cells to metastasize. The CD44 proteins carry amino acid sequence motifs that confer the ability to bind to the extracellular matrix component hyaluronate (HA). In this study, we investigated whether a CD44 variant previously shown to stimulate metastasis in a rat pancreatic carcinoma model (BSp73AS) is capable of binding to HA, and whether such binding is critical for metastasis. We show that transfection of this CD44 variant into BSp73AS cells increases the HA-binding capacity of the cells in a dose-dependent manner. Transfection of the same CD44 variant isoform into BDX2 cells also conferred strong HA-binding properties on these cells, but was insufficient to cause them to metastasize. Transfection of a surface-bound hyaluronidase into metastasizing BSp73AS cells bearing variant CD44 efficiently ablated the ability of these cells to bind to HA. However, in metastasis assays, these hyaluronidase-transfected cells showed patterns of metastasis similar to those of the parental cell line. We also show that the HA-binding capacity of a variety of tumor cells is not correlated with their metastatic proclivity, and that an antibody previously shown to block metastasis of the pancreatic carcinoma cells does not interfere with their ability to bind to HA. We conclude that although CD44 variant expression does promote metastasis formation, HA binding by tumor cells is not rate limiting for metastasis in the BSp73AS system and probably also in other metastasizing tumors. Furthermore, for metastasis by CD44 variant-expressing BSp73AS cells to occur, contact of the CD44 variant protein with a ligand other than HA Is required.

Increasing incidence of CD44v7/8 epitope expression during uterine cervical carcinogenesis.

Splice variants of the cell surface glycoprotein CD44 (CD44v) have been implicated in the progression of various human tumors. In the present study, we have examined the expression pattern of a CD44v epitope encoded by the adjacent variant exons v7 and v8 during human cervical carcinogenesis. While only l/ll normal cervical squamous epithelia was positive for this epitope by immunohistochemistry, 4/21 samples of low-grade squamous intra-epithelial lesions (LSIL), 17/35 samples of high-grade squamous intra-epithelial lesions (HSIL), 11/12 samples of the HSIL subgroup of carcinomas in situ and 17/17 cases of invasive cervical carcinoma showed CD44v7/8 epitope expression. In addition to CD44 variant expression, we have analyzed 67 lesions for the presence of HPV16/18-DNA using PCR. Most of the samples expressing the v7/8 epitope were also HPV16-positive (29/32), whereas only 17/35 of the v7/8-negative samples were HPV16-positive. HPV18 DNA was found in only one invasive carcinoma. Our data suggests that high-risk HPV infection may precede CD44v7/8 expression and that the number of samples expressing the CD44v7/8 epitope increases during carcinogenesis and reaches nearly 100% at the carcinoma in situ stage. This CD44 epitope could, therefore, serve as a diagnostic marker of cervical squamous cell carcinomas and as a possible target for CD44v7/8 epitope-directed therapies.

EGF receptor and p185erbB-2-specific single-chain antibody toxins differ in their cell-killing activity on tumor cells expressing both receptor proteins.

Many human tumors over-express erbB-2 and EGF receptors. The membrane localization of these receptor tyrosine kinases make them appropriate targets for directed tumor therapy. We have used recombinant DNA technology to produce single-chain antibody exotoxin A (scFv-ETA) fusion proteins which specifically bind the erbB-2 and EGF receptors. The scFv portion is composed of the heavy- and light-chain variable domains of monoclonal antibodies which recognize the extracellular portion of each receptor. We have previously described the anti-tumor activity of the bacterially produced scFv(FRP5)-ETA directed to the erbB-2 receptor. In this paper we describe the characteristics of scFv(225)-ETA, a protein which binds the EGF receptor. The bacterially produced recombinant protein binds to the receptor with high affinity and inhibits the in vitro growth of the EGF receptor over-expressing tumor cell lines A431 and MDA-MB468. Combination treatment with scFv-(FRP5)-ETA and scFv(225)-ETA led to an additive inhibitory effect on the in vitro growth of A431 cells. SKBR3 cells expressing low levels of EGF receptor but high levels of p185erbB-2 were not affected by scFv(225)-ETA treatment but were sensitive to scFv(FRP5)-ETA. Stimulation of SKBR3 cells and HCII RI#11 mouse mammary epithelial cells expressing the human erbB-2 with EGF led to an increase in scFv(FRP5)-ETA activity, showing that the EGF-induced activation of erbB-2 can potentiate the action of the erbB-2-directed toxin. Treatment of athymic nude mice with scFv(FRP5)-ETA and the combination of both scFv-ETA proteins led to the transient arrest of growth of established A431 tumors. scFv(225)-ETA treatment alone was the most effective, leading to tumor shrinkage during the course of treatment, whereas treatment with the parental monoclonal antibody 225 led to retarded tumor growth.

Surface protein expression and messenger RNA-splicing analysis of CD44 in uterine cervical cancer and normal cervical epithelium.

Variant CD44 has recently been shown to serve as a metastasis marker in human breast cancer. Certain variant epitopes on primary tumors predict poor survival probabilities for the patients. In this study, immunohistochemical analysis of 16 uterine cervical carcinomas showed strong expression of several CD44 variant epitopes in all samples. In normal cervical epithelia from 5 patients, expression of these epitopes was restricted to particular cell layers, with expression being strong in basal and spinal cells but absent in superficial cells. Fifteen of 16 cancer samples were stained strongly with an antibody which recognizes one particular CD44 epitope that is encoded by both variant exons v7 and v8. This epitope was not detectable in normal cervical epithelium. CD44-mRNA splicing analysis showed qualitative and quantitative differences between malignant and normal tissues with a much more complex splice pattern and high expression of a large CD44 isoform containing variant exons v3 to v10 (including the v7/v8 transition epitope) in about one-half of the cancer samples. Interestingly, patients with lymph node metastases were in this group only. These differences in CD44 epitope expression and mRNA splicing in cervical carcinoma reveal dynamic changes in CD44 expression during carcinogenesis. Such changes could provide metastatic cells with a selective advantage during the carcinogenic process. Furthermore, the v7/v8 epitope may be suitable for screening early stages of cervical cancer.

Interphase cytogenetic study of childhood acute lymphoblastic leukemia.

We used the fluorescence in situ hybridization (FISH) technique and centromere-specific probes for chromosomes 1, 6, 8, 10, 12, 17, 18, X, and Y to investigate the presence and number of the respective chromosomes in interphase nuclei of 14 cases of childhood acute lymphoblastic leukemia (ALL) which were shown to be hyperdiploid by DNA flow cytometry irrespective of their cytogenetic pattern. Numerical anomalies for one or more chromosomes were detected in all 14 cases. The FISH results were compared with those obtained by conventional cytogenetic analysis. A hyperdiploid karyotype was evident in 5 cases, the others were either normal or lacking cytogenetic results because of technical failure. In the 5 cytogenetically hyperdiploid cases, 14 numerical abnormalities were observed with both techniques, whereas 4 numerical deviations were found only with FISH. In 9 other cases which had a DNA content indicating hyperdiploidy, 34 trisomies and 2 tetrasomies were detected by FISH analysis. Furthermore, in 1 case duplication of the Y chromosome and in 3 male cases duplication of the X chromosome were evident. Double-target FISH experiments in 2 patients allowed the correlation of numerical aberrations of 2 chromosomes in one and the same cell. By such analyses, detection of subpopulations of tumor cells was found to be relatively easy. Our results indicate that the FISH technique with chromosome-specific repetitive centromeric probes is a rapid, simple to use, and easy to interpret technique for the evaluation of numerical chromosomal aberrations in interphase nuclei of leukemias.

Analysis of molecular functions of the tumor metastasis promoting surface molecule CD44v4-v7 using transgenic mice.

In certain circumstances, metastatic tumor cells may mimic the molecular properties and behaviour of lymphocytes. Support for this hypothesis has come from the observation that activated lymphocytes and some tumor cells need a CD44 variant isoform (CD44v) to survive and/or expand in the lymphatic system. CD44 variant (CD44v) isoforms are created by differential splicing from a pool of at least ten variant exons (v1-v10), the encoded sequences of which are absent in the CD44 standard isoform (CD44s). To dissect the molecular interactions of CD44v, transgenic animals have been generated that constitutively express a variant of CD44 containing sequences encoded by exons v4 to v7 on the surface of T cells. Lymphocytes derived from these transgenic animals show accelerated entry into S phase upon antigenic stimulation, and a subpopulation of the cells constitutively express early lymphocyte activation markers. Our data support the hypothesis that the presence of CD44v4-v7 on the surface of T cells mediates intercellular or intracellular processes which result in the promotion of T cells towards a preactivated state.