Activating transcription factor-2 in survival mechanisms in head and neck carcinoma cells.

BACKGROUND: Activating transcription factor-2 (ATF2) is associated with tumor progression but is not well studied in head and neck squamous cell carcinoma (HNSCC). Its effects in stress and its importance in other survival mechanisms were studied. METHODS: ATF2 expression and nuclear activation were confirmed in HNSCC. After modulation of ATF2, in vitro effects on proliferation and chemosensitivity were studied. Effects on in vivo tumor growth and interleukin 8 (IL-8) expression were determined. Tumor necrosis factor-alpha (TNF-α) treatment was used to further evaluate cytokine production and chemosensitivity. RESULTS: Reductions of ATF2 resulted in significant nuclear p-ATF2 activation, cisplatin resistance, and augmented IL-8 expression without affecting in vivo tumor growth. In this setting, TNF increases p-p38 phosphorylation and chemosensitivity while further enhancing IL-8 production. CONCLUSION: Our data suggest regulatory roles for ATF2 in TNF-related mechanisms of HNSCC. Its perturbation and nuclear activation are associated with significant effects on survival and cytokine production.

Genomic comparison of invasive and rare non-invasive strains reveals Porphyromonas gingivalis genetic polymorphisms.

BACKGROUND: Porphyromonas gingivalis strains are shown to invade human cells in vitro with different invasion efficiencies, varying by up to three orders of magnitude. OBJECTIVE: We tested the hypothesis that invasion-associated interstrain genomic polymorphisms are present in P. gingivalis and that putative invasion-associated genes can contribute to P. gingivalis invasion. DESIGN: Using an invasive (W83) and the only available non-invasive P. gingivalis strain (AJW4) and whole genome microarrays followed by two separate software tools, we carried out comparative genomic hybridization (CGH) analysis. RESULTS: We identified 68 annotated and 51 hypothetical open reading frames (ORFs) that are polymorphic between these strains. Among these are surface proteins, lipoproteins, capsular polysaccharide biosynthesis enzymes, regulatory and immunoreactive proteins, integrases, and transposases often with abnormal GC content and clustered on the chromosome. Amplification of selected ORFs was used to validate the approach and the selection. Eleven clinical strains were investigated for the presence of selected ORFs. The putative invasion-associated ORFs were present in 10 of the isolates. The invasion ability of three isogenic mutants, carrying deletions in PG0185, PG0186, and PG0982 was tested. The PG0185 (ragA) and PG0186 (ragB) mutants had 5.1×10(3)-fold and 3.6×10(3)-fold decreased in vitro invasion ability, respectively. CONCLUSION: The annotation of divergent ORFs suggests deficiency in multiple genes as a basis for P. gingivalis non-invasive phenotype.

Cultivation of Enterobacter hormaechei from human atherosclerotic tissue.

AIM: To determine whether culturable bacterial strains are present in human atheromatous tissue and to investigate their properties using culture, quantitative PCR, metagenomic screening, genomic and biochemical methods. METHODS: We analyzed femoral atherosclerotic plaque and five pairs of diseased and healthy arterial tissue for the presence of culturable bacteria using cell cultures and genomic analysis. RESULTS: Gram negative aerobic bacilli were cultivated from the plaque tissue. Ribosomal 16S DNA amplification and sequencing identified the isolates as Enterobacter hormaechei. The isolate was resistant to ampicillin, cefazolin, and erythromycin. A circular 10 kb plasmid was isolated from the strain. Antibiotic protection assays of the isolate demonstrated invasive ability in a human monocytic cell line. To extend the study, five matched pairs of diseased and healthy aortic tissue were analyzed via quantitative PCR. Eubacterial 16S rDNA was detected in all specimens, however, E. hormaechei DNA was detected in surprisingly high numbers in two of the diseased tissues only. CONCLUSIONS: While it is well documented that inflammation is an important risk factor for vascular pathophysiology, the association of bacteria with atherosclerosis has not been clearly established, in large part due to the inability to isolate live bacteria from atheromatous tissue. This is the first study providing direct evidence of Enterobacter spp. associated with atheromatous tissues. The data suggest that chronic infection with bacteria may be an under-reported etiologic factor in vascular pathogenesis. Importantly, characterization of the clinical isolate supports a model of atherogenesis where systemic dissemination of bacteria to atherosclerotic sites may occur via internalization in phagocytic cells.

Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption.

We have evaluated the role of the ADP-ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+-releasing second messenger, cyclic ADP-ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin-6 (IL-6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38-/- mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38-/- mice showed a dramatic approximately fourfold increase in osteoclast formation in response to incubation for 6 days with RANK-L and M-CSF. The osteoclasts so formed in these cultures showed a approximately 2.5-fold increase in resorptive activity compared with wild-type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase-positive colony-forming units (CFU-Fs), those derived from CD38-/- mice showed a significant reduction in differentiation compared with wild-type cells. Real-time RT-PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL-6 and IL-6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, alpha, beta, and gamma. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions as a cellular NAD+ "sensor," particularly during periods of active motility and secretion.

Transduction of TAT fusion proteins into osteoclasts and osteoblasts.

It has been difficult to transduce primary cultures of bone cells with proteins of interest. Here, we report the development and validation of a new technology for transduction of osteoblasts and osteoclasts with peptides and moderately sized proteins. Fusion proteins between TAT, an 11 amino acid Arg-rich sequence derived from the HIV protein, and either hemagglutinin or calcineurin Aalpha were synthesized and purified. Exposure of osteoblasts and osteoclasts in primary culture to either TAT-HA or TAT-calcineurin Aalpha resulted in a rapid (within 10 min) intracellular movement of the fusion protein evident on co-immunostaining. Almost 99% of cells were transduced and the fusion protein was retained in approximately 50% of the cells for up to 5 days. TAT did not abolish the functionality of calcineurin Aalpha; the fusion protein stimulated osteoblast differentiation and inhibited osteoclastic resorption. We expect that our studies will provide a firm basis for the future development of TAT fusion proteins for critical molecules involved in bone cell differentiation and function.