Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease.

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.

Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: establishment of working models for human micrometastases.

Bone marrow (BM) is a clinically relevant site of micrometastatic disease in patients with solid epithelial tumors. It is, therefore, important to establish suitable models that allow the in-depth characterization of disseminated tumor cells present at low frequencies of 10(-5)-10(-6) nucleated BM cells. The aim of this study was to assess common phenotypic features of nine tumor cell lines established from BM of patients with cancer of the prostate (four cell lines), breast (two cell lines), lung (two cell lines), and colon (one cell line) using immunocytochemistry, flow cytometry, and reverse transcription-PCR. All cell lines stained positive for both cytokeratins, the epithelial intermediate filaments, and the epithelial cell adhesion molecule E-cadherin, and they lacked markers of BM-derived cells. The tumor origin of the cell lines was supported by the expression of the ErbB2 oncogene (seven of nine) and MAGE mRNA (eight of eight). All cell lines coexpressed cytokeratin and vimentin, the mesenchymal intermediate filament, indicating an epithelial-mesenchymal transition of micrometastatic cells. The invasive phenotype of the immortalized cells was also reflected by the consistent expression of several metastasis-associated adhesion molecules, including alpha5 (eight of nine), alpha6 (five of nine), alphaV (nine of nine), beta1 (nine of nine), and beta3 (nine of nine) integrin subunits and the Mr 67,000 laminin receptor (seven of nine). Contrary to our expectations, metastasis-promoting CD44 variant isoforms were only detected on two lines, whereas all cell lines expressed MUC18/melanoma cell adhesion molecule and intercellular adhesion molecule-1, two members of the immunoglobulin superfamily of adhesion molecules that are not frequently found on primary carcinoma cells. The consistent expression of various epithelial and tumor-associated antigens provides evidence that the established cell lines are derived from disseminated cancer cells present in the BM. The invasive phenotype of the immortalized cells was mirrored by their epithelial-mesenchymal transition and the expression of several metastasis-associated molecules, which might be potential candidates for novel therapeutic targets.

Three different but related gene clusters encoding gas vesicles in halophilic archaea.

We present an analysis of the chromosomal region comprising the gene cluster involved in gas vesicle (Vac) synthesis in Haloferax mediterranei (mc-vac-region) and Halobacterium salinarium (c-vac-region) and compare both of them to the plasmid located p-vac-region of H. salinarium. The p-vac-region of 9000 base-pairs (9 kb) is more related to mc-vac (9.4 kb) of Hf. mediterranei than it is to the c-vac-region (8.3 kb) present in the same cell. The Vac- species Hf. volcanii becomes Vac+ following transformation with a fragment containing the entire mc-vac-region. Also the p-vac-region transforms Hf. volcanii to a Vac+ phenotype, indicating that this gene cluster is sufficient for gas vesicle synthesis and does not depend on products of the c-vac-region. Each of these vac-regions contains, in addition to gvpA encoding the major gas vesicle protein, 13 open reading frames named gvpC through gvpO. Ten of these, gvpD through gvpM, are located upstream from gvpA in opposite orientation, while gvpC, gvpN and gvpO are found 3' to gvpA. The absolute requirement of gvpO for gas vesicle synthesis was demonstrated by transformation experiments. Northern analyses with RNA samples isolated during the growth cycle of Hf. mediterranei or of H. salinarium PHH4 revealed that the mc-gvpD or c-gvpD mRNAs occur similar to the respective gvpA mRNA in stationary growth phase, while gvpF-gvpM are transcribed mainly during logarithmic growth. S1-nuclease mapping was performed to determine the transcriptional start site of the gvpD mRNA. The distance between the two divergent start sites of gvpA and gvpD mRNA is 109 base-pairs in mc-vac and p-vac, while in the case of c-vac this distance is 22 base-pairs larger. The conservation of the various gvp products, characteristic features and their possible functions in gas vesicle synthesis are discussed.

Commentary. An uninformed choice.

Judaism is wrongly blamed for exacerbating the dilemma facing this Orthodox patient and her family. In actuality, a vital and human halakhic tradition is a resource that can respect the daughter's autonomy while appropriately responding to her father's fears about her well-being.

Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-gvpDE genes.

Gas-vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p-vac region consisting of 14 different gvp genes that are arranged in two clusters: p-gvpACNO and, oppositely oriented, p-gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p-gvpDE and p-gvpF-M. The 5'-terminus of the p-gvpF-M mRNA was located 169 nucleotides upstream of p-gvpF within p-gvpE. The p-gvpG and p-gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas-vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p-gvp gene expression for gas-vesicle synthesis was determined by transformation experiments using the Vac- species Haloferax volcanii as recipient. Construct delta A containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac- transformants, but the addition of p-gvpA on a second vector restored gas-vesicle synthesis to wild-type level (Vac++). Similarly, double transformants containing p-gvpD-M plus p-gvpACNO, or p-gvpG-M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p-gvpFED-ACNO were Vac++. Transformants containing the p-vac region either lacking gvpA, gvpF, or gvpGHI were Vac-, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas-vesicle formation. Double transformants containing the constructs p-gvpF-M plus p-gvpACNO (delta DE) accumulated gas vesicles (Vac+) but synthesized fewer than the wild type, showing that the p-gvpDE genes are not necessary for gas-vesicle assembly. A repressor function affecting the synthesis of the p-gvpF-M mRNA could be suggested for p-gvpD and the 5'-region of its mRNA.

Functional studies of the gvpACNO operon of Halobacterium salinarium reveal that the GvpC protein shapes gas vesicles.

Gas vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the plasmid pHH1-encoded vac (p-vac) region consisting of 14 different gvp genes that are arranged in two clusters, p-gvpACNO and, oriented in the direction opposite to that of gvpA, p-gvpDEFGHIJKLM. The p-gvpACNO region was analyzed at the transcriptional and functional levels in H. salinarium and in Haloferax volcanii transformants containing subfragments of the p-vac region. The p-gvpACNO genes were transcribed as several mRNAs: the 270-nucleotide (nt) p-gvpA transcript, encoding the major structural protein, occurred in large amounts, and minor amounts of three different readthrough transcripts (p-gvpACN, and p-gvpACNO mRNA) were found. In addition, the p-gvpO gene gave rise to two separate mRNA species: a 550-nt mRNA starting at the ATG and spanning the entire reading frame and a 420-nt RNA encompassing the second half of the p-gvpO gene. The requirement of p-gvpC, p-gvpN, and p-gvpO gene expression for gas vesicle synthesis was assessed by transformation experiments using the VAC- species Haloferax volcanii as the recipient. A delta C transformant, harboring the p-vac region with a deletion of the p-gvpC gene, produced large amounts of irregularly shaped gas vesicles. A shape-forming function of p-GvpC was demonstrated by complementation of the delta C transformant with the p-gvpC gene, resulting in wild-type-shaped gas vesicles. In the delta N transformant, the level of gas vesicle synthesis was very low, indicating that the p-GvpN protein is not required for gas vesicle assembly but may enhance gas vesicle synthesis. The p-gvpN deletion did not affect accumulation of p-gvpACO mRNA but reduced the separate p-gvpO transcription. The delta O transformant was Vac- and had a strongly decreased level of p-gvpACN mRNAs, demonstrating that the p-GvpO protein is required for gas vesicle synthesis and may affect transcription of this DNA region.

Eight of fourteen gvp genes are sufficient for formation of gas vesicles in halophilic archaea.

The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined. Single genes of the 14 gvp genes present in the p-vac region on plasmid pHH1 of Halobacterium salinarum (p-gvpACNO and p-gvpDEFGHIJKLM) were deleted, and the remaining genes were tested for the formation of gas vesicles in Haloferax volcanii transformants. The deletion of six gvp genes (p-gvpCN, p-gvpDE, and p-gvpHI) still enabled the production of gas vesicles in H. volcanii. The gas vesicles formed in some of these gvp gene deletion transformants were altered in shape (Delta I, Delta C) or strength (Delta H) but still functioned as flotation devices. A minimal p-vac region (minvac) containing the eight remaining genes (gvpFGJKLM-gvpAO) was constructed and tested for gas vesicle formation in H. volcanii. The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy. Transcript analyses demonstrated that minvac transformants synthesized regular amounts of gvpA mRNA, but the transcripts derived from gvpFGJKLM were mainly short and encompassed only gvpFG(J), suggesting that the gvpJKLM genes were not sufficiently expressed. Since gvpAO and gvpFGJKLM are the only gvp genes present in minvac/JKLM transformants containing gas vesicles, these gvp genes represent the minimal set required for gas vesicle formation in halophilic archaea. Homologs of six of these gvp genes are found in Anabaena flos-aquae, and homologs of all eight minimal halobacterial gvp genes are present in Bacillus megaterium and in the genome of Streptomyces coelicolor.

Gas vesicle formation in halophilic Archaea.

Gas vesicles are intracellular, microbial flotation devices that consist of mainly one protein, GvpA. The formation of halobacterial gas vesicles occurs along a complex pathway involving 14 different gvp genes that are clustered in a genomic region termed the "vac region". Various vac regions found in Halobacterium salinarum (p-vac and c-vac), Haloferax mediterranei (mc-vac), and Natronobacterium vacuolatum (nv-vac) have been investigated. Except for the latter vac region, the arrangement of the gvp genes is identical. Single gvp genes have been mutated to study the effect on gas vesicle synthesis in transformants and to determine their possible function. Each vac region exhibits a characteristic transcription pattern, and regulatory steps have been observed at the DNA, RNA, and protein level, indicating a complex regulatory network acting during gas vesicle gene expression.

p53 gene mutations are not required for early dissemination of cancer cells.

The p53 protein is involved in several central cellular processes, including gene transcription, DNA repair, cell cycling, genomic stability, chromosomal segregation, senescence, and apoptosis. p53 mutations frequently result in an immunocytochemically detectable accumulation of the p53 protein in tumor cells. To evaluate whether p53 gene mutations are required for the onset of hematogeneous tumor cell dissemination, we compared the p53 status of primary and micrometastatic tumor cells. Disseminated carcinoma cells could be detected in bone marrow aspirates obtained from 46 (40%) of 114 patients with various types of epithelial tumors without overt skeleton metastases. There was no correlation between the detection of p53 protein in primary lung carcinomas and the presence of tumor cells in bone marrow. Further analyses revealed that the disseminated carcinoma cells rarely accumulate mutated p53 protein and that 10 cell lines derived thereof did not harbor p53 mutations even in the presence of such mutations in the autologous primary tumors. These observations indicate that tumor cells can leave the primary tumor before mutations of the p53 gene occur and that these mutations are not essential for such early hematogeneous dissemination of cancer cells. Thus, the value of mutated p53 as a target for diagnosis and treatment of micrometastatic disease in cancer patients is questionable.