The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

Effects of endostatin production on oncogenicity and metastatic activity of HPV16-transformed mouse cells: role of interleukin 1alpha.

Two mouse HPV16-transformed cell lines, viz. MK16 cells, which induce metastasizing tumors, and TC-1 cells, which induce non-metastasizing tumors were transduced with the gene for mouse endostatin. Two clones constitutively expressing endostatin were isolated from each of them. They were denoted ME3 and ME9, and TE2 and TE5, respectively. When inoculated into mice, ME3 cells were non-oncogenic. Nearly all mice inoculated with ME9 cells developed tumors, but considerably later than did the parental MK16 cells and metastasis formation was strongly reduced in these animals. On the other hand, TE2 and TE5 cells displayed oncogenic potential similar to that of the parental cells. To provide more information on these different effects of endostatin production, cell lysates of all six lines studied were tested for the content of 25 factors known to be involved in angiogenesis. The parental MK16 cells differed from the parental TC-1 cells and also from all endostatin producing sublines by a markedly higher production of interleukin 1alpha (IL-1alpha) and, to a lesser extent, by a higher production of several other factors tested. Additional experiments indicated that the suppression of the production of IL-1alpha by the parental MK16 caused by endostatin was due to an autocrine mechanism.

Live cell vaccines expressing B7.1, monocyte chemoattractant protein 1 and granulocyte-macrophage colony stimulation factor derived from mouse HPV16-transformed cells.

One of the gene therapy strategies in oncology is immunization with cancer cells that express various cytokines. We used a thymidine-kinase deficient (cTK-) cell line designated 123IA, which had been derived from HPV16-transformed mouse (C57BL/6) cells MK16/I/III/ABC (MK16). To obtain genetically modified cells, 123IA cells were transfected with bicistronic plasmid vectors carrying the herpes simplex type 1 thymidine kinase (HSV TK) gene and either the gene for the mouse B7.1 (CD80) co-stimulatory molecule or the gene for the monocyte-chemoattractant protein 1 (MCP-1). For control purposes, a plasmid vector carrying only the HSV TK gene was used. The transfected cells were cultivated in medium supplemented with hypoxanthine, aminopterin and thymidine. For comparative purposes we also used B9 cells, which express the granulocyte-macrophage colony stimulation factor (GM-CSF) and had been derived from 123A cells by transduction with the recombinant adeno-associated virus carrying the HSV TK gene and the mouse GM-CSF gene. All of the cell lines isolated were found to be sensitive to minute amounts of ganciclovir, revealing the production of HSV TK, and to express the respective transgenes. When inoculated into 5-week-old female syngeneic mice, cells expressing either GM-CSF or B7.1 were non-oncogenic. On the other hand, nearly all mice inoculated with MCP-1-producing cells developed tumours, though considerably later than animals inoculated with the same dose of the parental MK16 cells. Animals injected with GM-CSF- or B7.1-producing cells were protected against challenge with the parental MK16 cells. When another mouse (C57BL/6) HPV16-transformed oncogenic cell line, TC-1, which differs from the MK16 cells in a number of properties such as MHC class I and B7.1 expression, was used for the challenge, the protective effect was much less pronounced.

Immunization with live HPV-16-transformed mouse cells expressing the herpes simplex thymidine kinase and either GM-CSF or IL-2.

From mouse (C57BL/6) HPV-16 transformed cells denoted MK16/1/IIIABC (MK16) a cellular thymidine kinase deficient (cTK-) cell line was isolated. These cTK- cells were transduced by bicistronic recombinant adeno-associated viruses (rAAV) carrying the herpes simplex virus thymidine kinase gene and the gene for either the mouse granulocyte-macrophage colony stimulating factor (GM-CSF) or mouse interleukin-2 (IL-2). Transduced cells were highly sensitive to minute amounts of ganciclovir (GCV) and synthesized moderate amounts of the respective cytokines. A number of cell clones were tested for the cytokine production. The two best producer cell lines, the GM-CSF-producing cells denoted B9 and the IL-2-producing cells denoted 181, were selected for further experiments. Neither B9 nor 181 cells were tumorigenic in syngeneic animals. As inducers of antitumour immunity against challenge with MK16 cells, B9 cells proved superior to the 181 cells. GCV treatment did not markedly influence the level of immunity induced.