Modification of proto-oncogene expression by phorbol esters in human dermal microvascular endothelial cells.

Following activation with the inflammatory mediator phorbol myristate acetate (PMA), human microvascular endothelial cells (DMEC) is olated from the human dermis (DMEC) rapidly and dramatically convert from a classical epithelioid morphology to a spindle-shaped configuration. This is accompanied by changes in the organization of gap junctions and the vimentin and actin cytoskeletons. This report describes the sequential changes in the expression of four proto-oncogenes, c-fos, c-myc, c-sis and H-ras in DMEC following PMA exposure. The synthesis of c-fos mRNA was transiently induced by PMA from a basal concentration below the limit of detection to a maximum at 60 min., declining to the unstimulated level within 2 hrs. Synthesis of c-myc mRNA declined continuously and reached 37% of control levels over 16 hrs. Expression of c-sis which encodes for the B chain of platelet-derived growth factor, also declined to 34% of the control value over 16 hrs. There was no change in the synthesis of H-ras mRNA nor of beta-actin mRNA which was used as a control. The expression of c-myc in normal DMEC was compared to a human dermal microvascular cell line transformed by SV-40 (TREND). The TREND cell line maintains a permanent spindle-shaped configuration under all growth conditions and multiplies faster than DMEC. In contrast to the non-transformed cell cultures, expression of c-myc in TREND cells was induced by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of attachment and growth of tumor cells on collagen by a monoclonal antibody

Summary A murine monoclonal antibody, VM-1, which binds to basal cells of normal human epidermis, reduces the ability of human squamous cell carcinoma cells (SCL-1) derived from the skin to attach and spread on collagen by about 50% and causes cell rounding. Similar effects have been previously shown using normal human keratinocytes. The attachment of cell lines derived from human lung squamous cell carcinomas (SW1271 and SW900), melanoma A375, glioblastoma 126, and fibrosarcoma HT1080 is also inhibited by this antibody. VM-1 antibody does not bind to normal human fibroblasts, benign nevus cells, or the human B-cell-derived line 8866. VM-1 antibody inhibits the growth of SCL-1 cells in vitro as measured by cell numbers and [³H]thymidine ([³H]TdR) incorporation. It is not cytolytic in the presence of complement as measured by⁵¹Cr release. Repeated treatment of SCL-1 cells with VM-1 antibody significantly reduces the proportion of SCL-1 cells that attach to collagen. In addition, after treatment of SCL-1 cells with VM-1 antibody, several proteins can no longer be demonstrated by gel electrophoresis of the cell-free supernatant. The VM-1 antibody effect on attachment and spreading is partially reversed by pretreatment of the collagen surface with laminin and fibronectin, but not with the carbohydrates chondroitin-6-sulfate or hyaluronic acid or with the protein lysozyme. By fluorescence staining, the antigen recognized by VM-1 antibody is membrane-bound and Triton X-100 extractable. The VM-1 antigen is excluded from Bio-Sil TSK-400 and sediments at about 10.5 S. It has a covalent molecular weight on the order of 10⁶. Proteinase K digestion produces VM-1 antibody reactive fragments, assumed to be polysaccharides, with a polydisperse molecular weight distribution in the range 5000 to 30 000. The VM-1 antigen is partially lost from solution on boiling and is no longer detectable in the aqueous or organic phase after chloroform-methanol extraction. The properties of the VM-1 antigen are consistent with those of a proteoglycan involved in attachment and spreading of kerationcytes and certain tumor cells on collagen. This research was supported by a grant from the Elsa U. Pardee Foundation, a Training Grant from the National Institutes of Health, Bethesda, MD, and the Psoriasis Research Institute. Part of this work has appeared as an abstract in Fed. Proc. 43:1929, Abst. #2994, 1984.