35 research outputs found
Structurally specific thermal fluctuations identify functional sites for DNA transcription
We report results showing that thermally-induced openings of double stranded
DNA coincide with the location of functionally relevant sites for
transcription. Investigating both viral and bacterial DNA gene promoter
segments, we found that the most probable opening occurs at the transcription
start site. Minor openings appear to be related to other regulatory sites. Our
results suggest that coherent thermal fluctuations play an important role in
the initiation of transcription. Essential elements of the dynamics, in
addition to sequence specificity, are nonlinearity and entropy, provided by
local base-pair constraints
Human Sulfatase 2 inhibits in vivo tumor growth of MDA-MB-231 human breast cancer xenografts
BACKGROUND: Extracellular human sulfatases modulate growth factor signaling by alteration of the heparin/heparan sulfate proteoglycan (HSPG) 6-O-sulfation state. HSPGs bind to numerous growth factor ligands including fibroblast growth factors (FGF), epidermal growth factors (EGF), and vascular endothelial growth factors (VEGF), and are critically important in the context of cancer cell growth, invasion, and metastasis. We hypothesized that sulfatase activity in the tumor microenvironment would regulate tumor growth in vivo. METHODS: We established a model of stable expression of sulfatases in the human breast cancer cell line MDA-MB-231 and purified recombinant human Sulfatase 2 (rhSulf2) for exogenous administration. In vitro studies were performed to measure effects on breast cancer cell invasion and proliferation, and groups were statistically compared using Student's t-test. The effects of hSulf2 on tumor progression were tested using in vivo xenografts with two methods. First, MDA-MB-231 cells stably expressing hSulf1, hSulf2, or both hSulf1/hSulf2 were grown as xenografts and the resulting tumor growth and vascularization was compared to controls. Secondly, wild type MDA-MB-231 xenografts were treated by short-term intratumoral injection with rhSulf2 or vehicle during tumor growth. Ultrasound analysis was also used to complement caliper measurement to monitor tumor growth. In vivo studies were statistically analyzed using Student's t test. RESULTS: In vitro, stable expression of hSulf2 or administration of rhSulf2 in breast cancer cells decreased cell proliferation and invasion, corresponding to an inhibition of ERK activation. Stable expression of the sulfatases in xenografts significantly suppressed tumor growth, with complete regression of tumors expressing both hSulf1 and hSulf2 and significantly smaller tumor volumes in groups expressing hSulf1 or hSulf2 compared to control xenografts. Despite significant suppression of tumor volume, sulfatases did not affect vascular density within the tumors. By contrast, transient exogenous treatment of MDA-MB-231 xenografts with rhSulf2 was not sufficient to inhibit or reverse tumor growth. CONCLUSION: These data indicate that in vivo progression of human breast cancer xenografts can be inhibited with sulfatase expression, and therapeutic effect requires constant delivery at the tumor site. Our results support a direct effect of sulfatases on tumor growth or invasion, rather than an effect in the stromal compartment
Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases
Background: Complement C2 deficiency is the most common genetically determined complete complement deficiency and is associated with a number of diseases. Most prominent are the associations with recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults. The links with these diseases reflect the important role complement C2 plays in both innate immunity and immune tolerance. Infusions with normal fresh frozen plasma for the treatment of associated disease have demonstrated therapeutic effects but so far protein replacement therapy has not been evaluated. Results: Human complement C2 was cloned and expressed in a mammalian cell line. The purity of recombinant human C2 (rhC2) was greater than 95% and it was characterized for stability and activity. It was sensitive to C1s cleavage and restored classical complement pathway activity in C2-deficient serum both in a complement activation ELISA and a hemolytic assay. Furthermore, rhC2 could increase C3 fragment deposition on the human pathogen Streptococcus pneumoniae in C2-deficient serum to levels equal to those with normal serum. Conclusions: Taken together these data suggest that recombinant human C2 can restore classical complement pathway activity and may serve as a potential therapeutic for recurring bacterial infections or SLE in C2-deficient patients
Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases
The adenovirus major late promoter TATA box and initiation site are both necessary for transcription in vitro.
Mutagenized DNA templates and HeLa whole cell extracts were used to study the effects of promoter-specific base changes on in vitro transcription. DNA templates where the initiating adenine (+1) was changed to thymidine (AT+1) in the adenovirus 2 major late transcription unit were transcribed with 50% efficiency of the unaltered template. We have described a mutant at the TATA box, where the A at position -28 was changed to a C (AC-28). Transcription efficiency was reduced to less than 20% of control in the AC-28 mutant (Concino et al., 1983, J. Biol. Chem. 258: 8493-8496). Primer extension analysis revealed increased 5' end heterogeneity for in vitro transcripts derived from AC-28 and AT+1 DNA templates. Specific transcription was completely abolished from AT+1 DNA templates when a second change was introduced within the TATA sequence, creating a double mutant (AC-28 . AT+1). Neither the AC-28 nor the AT+1 change alone had such an effect, suggesting a coordinated interaction in transcription initiation involving both the TATA box and the initiation site