CD44 expression and regulation during mammary gland development and function.

The CD44v6 epitope has been widely reported to be expressed in human mammary carcinomas, yet its prognostic significance is controversial and its function in mammary tumors and mammary glands is unknown. To begin to resolve these issues, we analysed in detail the normal postnatal expression patterns and regulation of the CD44v6 epitope in murine mammary glands. We demonstrate that significant CD44v6 epitope expression is first seen during puberty, and that after puberty CD44v6 epitope expression follows the estrous cycle. CD44v6 epitope expression is observed in the myoepithelium and also less widely in luminal epithelial cells. During lactation, CD44v6 epitope expression is turned off and reappears during involution. The CD44 variant isoform bearing the v6 epitope is CD44v1-v10. Using HC11, a mammary epithelial cell line with stem cell characteristics, and facilitated by the cloning of the murine CD44 promoter, we show that growth factors and hormones which regulate ductal growth and differentiation modulate CD44 transcription. Together our data suggest that the CD44v6 epitope is expressed in mammary epithelial stems cells and in lineages derived from these cells, and that CD44v6 expression is regulated in part by hormones and growth factors such as IGF-1 and EGF which regulate the growth and differentiation of the mammary epithelium. The function of these same growth factors and hormones is often perturbed in mammary carcinomas, and we suggest that CD44v6 expression in tumors reflects this perturbation. We conclude that the expression of the CD44v6 epitope observed in some mammary tumors reflects the stem cell origin of breast tumors, and that whether or not the CD44v6 epitope is expressed in a mammary tumor is determined by the differentiation status of the tumor cells.

Involvement of CD44 variant isoform v10 in progenitor cell adhesion and maturation.

CD44 has been described repeatedly to be involved in hematopoiesis. Here, we addressed the question of functional activity of CD44 variant isoform v10 (CD44v10) in progenitor cell maturation by in vivo and in vitro blocking studies with a monoclonal antibody and a receptor globulin. We became interested in this question by the observation that CD44v10 is expressed, although at a low level, on a subpopulation of bone marrow cells. Flow cytometry revealed that 15%-20% of hematopoietic cells in the fetal liver and 25%-35% of bone marrow cells in adult mice were CD44v10 positive. The majority of CD44v10+ cells was HSA+/J11d+ and CD43+. CD44v10 was not detected on CD4+, CD8+, IgM+, or IgD+ cells. A CD44v10 receptor globulin did not bind to hematopoietic progenitor cells, but to stromal elements. The CD44v10-CD44v10 ligand interaction had a major impact on the adhesion of progenitor cells to stromal elements. When healthy animals received repeated injections of either anti CD44v10 or the CD44v10 receptor globulin, committed progenitors were mobilized and significantly augmented numbers were recovered in the spleen and the peripheral blood. Furthermore, the CD44v10-CD44v10 ligand interaction, which had no impact on progenitor expansion, influenced progenitor maturation, particularly of the B-cell lineage. Although the nature of the CD44v10 ligand remains to be explored, the supportive role of CD44v10 in progenitor maturation and, importantly, the efficient mobilization of progenitor cells by anti-CD44v10 and a CD44v10 receptor globulin could be of clinical benefit in peripheral blood stem cell transplantation.

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.

Stable expression of a retrovirally transferred adhesion molecule in a human melanoma-specific cytotoxic T lymohocyte clone.

The adoptive transfer of in vitro generated tumor-specific cytotoxic T lymphocytes (CTL) is considered a promising perspective in cancer therapy. One possible drawback lies in the inappropriate homing of in vitro cultured lymphocytes, which could be circumvented by introducing the appropriate targeting molecules. Here we describe a protocol that allows a rapid and stable transfection of cytotoxic T cell clones. As a model system we used a CTL clone specific for the melanoma-associated antigen gp100 and a cDNA encoding for murine CD14 containing the variant exen v10 which is supposed to facilitate lymphocyte homing towards the skin. CD44v10 cDNA was ligated into the retroviral vector pMV-7, which was used to transfect the ecotropic GP-E-86 and the amphotropic PA317 cells. After several cycles of transduction to increase the viral titre, supernatants of the amphotropic PA317-CD44v10 line were used for transduction of CD44v10 into a human CTL clone. After three cycles of transduction at 12-h intervals, low but stable expression of CD44v10 was observed throughout the culture period of 10 weeks. The phenotype of the transduced CTL clone was unaltered and the cytotoxic potential was only slightly reduced as compared to the parental clone. The efficiency of stable transduction within a period of 1 week makes the protocol well suited for the in vivo transfer of transduced cells and, in the special case, should guarantee appropriate homing of the transduced CTL clone.

A link between ras and metastatic behavior of tumor cells: ras induces CD44 promoter activity and leads to low-level expression of metastasis-specific variants of CD44 in CREF cells.

The activated oncogene c-Ha-ras induces expression of the surface glycoprotein CD44 in cloned rat embryonic fibroblasts (CREF). Induction is transcriptional as shown by transient cotransfections of c-Ha-ras expression constructs and CD44 promoter reporter gene constructs and depends on the presence of an AP-1 binding site at position -110. Increased transcript levels for the standard isoform of CD44 (CD44s) are accompanied by the appearance of alternatively spliced RNAs and the synthesis of variants of CD44 (CD44v). These CD44v molecules differ from the standard type by the addition of sequences in the extracellular portion of the molecules. The occurrence of CD44v molecules in CREF cells upon induction of the CD44 promoter is probably due to leakiness of the splice control in these cells since stable transfection with c-Ha-ras does not alter the CD44v/total CD44 ratio. Upon ras overexpression, however, using an inducible mouse mammary tumor virus-ras construct, a transient increase of CD44v/total CD44 ratio of 3-4 has been determined suggesting that a burst of ras expression, in the genetic background of CREF cells, influences both promoter activity and splice control or accuracy. The expression of CD44v proteins is responsible for the metastatic potential in a variety of tumors (U. Günthert et al., Cell, 65: 13-24, 1991). Also in CREF cells expression of CD44v correlates with metastatic behavior, ras-transfected CREF cells are not only fully transformed but also give rise to metastatic spread as measured in the spontaneous metastasis assay. The adenoviral oncogene E1A counteracts ras-induced promoter function and, consequently, inhibits metastatic behavior without extinguishing transformation.