G. Arcidiaco, L. Belloni. F. Bevilacqua, G. Bruni, V. Cinquini, M. T. De Luca, G. Fenaroli, E. Giordano, G. P. Guidetti, M. La Forgia, M. A. Penco, O. Rossi, G. Tagliaferri, C. Tarsitani, J. Teichmann e P. Tucci—Storia della Fisica

Levi, F. A.

doi:10.1007/bf02813517

Cited by 6 publications

(8 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After initiation of osteogenesis, a number of other gene products, such as the zinc-finger transcription factors osterix and Krox-20, the homeobox-containing transcription factors, Msx2 and Dlx5, and various members of the Fos family members of transcription factors (c-fos, FosB, ⌬-FosB, Fra-1, and Fra-2), have been shown to influence the maintenance and regulation of osteoblastic differentiation. (44)(45)(46) Many of these molecules have also been implicated as having functional roles in both cell growth and cellular differentiation mediated through the actions of various TGF␤ superfamily members and fibroblast growth factors (FGFs). (47)(48)(49) A recent study by Takakura et al (50) showed that activator protein-1 (AP-1) was capable of repressing telomerase activity in a human cancer cell line through interaction of the AP-1 elements c-jun, JunD, and c-fos with the hTERT promoter.…”

Section: Discussionmentioning

confidence: 99%

hTERT Transcription Is Repressed by Cbfa1 in Human Mesenchymal Stem Cell Populations

Isenmann

Cakouros

Zannettino

et al. 2007

Journal of Bone and Mineral Research

View full text Add to dashboard Cite

Human BMSSCs lose telomerase activity in vitro, which leads to chromosomal instability and cellular senescence. We observed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. We showed that Cbfa1 acts as a partial repressor of TERT, which may facilitate cellular differentiation. Introduction:The absence of telomerase activity by cultured human bone marrow stromal stem cells (BMSSCs) causes critical shortening of chromosomal telomeres, leading eventually to cellular senescence. Ex vivo expansion of BMSSCs correlates to an increase in osteogenic lineage associated markers such as alkaline phosphatase, bone sialoprotein, and osteocalcin that are regulated by the master regulatory transcription factor, Cbfa1 (Runx2). This study examined whether Cbfa1 was capable of regulating the promoter of the early stem cell-associated gene, telomerase reverse transcriptase (TERT). Materials and Methods: Human BMSSCs were isolated by fluorescence-activated cell sorting. Telomerase activity was determined using the telometric repeat amplification protocol. CBFA1 and TERT gene expression was assessed by real-time PCR. The functional capacity of Cbfa1 to bind to the hTERT promoter was performed using a modified electrophoretic mobility shift assay (EMSA). Chromatin immunoprecipitation (ChIP) analysis was used to examine Cbfa1 binding to the hTERT promoter in vivo. Functional analysis of CBFA-1 wildtype and mutant DNA binding sites on TERT promoter fragments was assessed using the promoterless green fluorescence protein (GFP) reporter vector, pEGFP-1, after transfection into HOS cells. Results: This study showed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. The data showed that BMSSCs undergo osteogenic commitment after the loss of hTERT expression, with concomitant elevated levels of CBFA1 transcripts. In addition, two unique Cbfa1 DNA binding sites were identified on the hTERT proximal promoter by EMSA supershift assay. Mutated forms of the putative Cbfa1 binding sites, created by site-directed mutagenesis, were able to abolish this interaction. ChIP analysis showed that Cbfa1 interacted directly with the hTERT promoter in vivo. Functional studies using GFP reporter constructs, driven by 2-and 3-kbp hTERT proximal promoter fragments, showed significantly lower levels of transcriptional activity compared with corresponding constructs with mutated Cbfa1 binding site Oligo 2. Conclusions: These studies suggest that Cbfa1 may act as a repressor of early stem cell markers such as hTERT as one possible mechanism for facilitating cellular differentiation.

show abstract

Section: Discussionmentioning

confidence: 99%

hTERT Transcription Is Repressed by Cbfa1 in Human Mesenchymal Stem Cell Populations

Isenmann

Cakouros

Zannettino

et al. 2007

Journal of Bone and Mineral Research

View full text Add to dashboard Cite

show abstract

“…Thermal denaturation was performed by incubating Ca 2 pump solubilized in reconstitution bu¡er at 44³C. In a previous paper, we showed that under these conditions, thermal inactivation takes place through a common mechanism in the range 33^45³C that is well described by an exponential function with a lifetime of 2.5 h at 44³C [10].…”

Section: Selective Unfolding Of Speci¢c Domains Of the Ca 2+ Pumpmentioning

confidence: 99%

“…Thermal denaturation studies on the Ca 2 pump have demonstrated to be useful for revealing structural features of this protein [10]. Throughout this work, we employ this approach combined with £uorescence resonance energy transfer (FRET) measurements to make a close examination of the structural basis of the Ca 2 pump dimerization.…”

Section: Introductionmentioning

confidence: 99%

Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability

et al. 2000

View full text Add to dashboard Cite

Ca2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self-association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)-binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM-binding domains could be related with the previously found C28W-binding regions. ß

show abstract

“…The most rapid growth of implanted tumors in the SRC assay occurred after day 6, both in mice with syngeneic rodent tumors and in GSA-treated mice with human tumors. These results suggested that statistical discrimination of experimental treatments would be much improved [6][7][8][9][10][11][12] Slowly growing primary human tumor specimens from clinical biopsies may require longer growth periods (>10 days) for optimal discrimination of antitumor-drug activity, and the SRG assay in GSA-treated mice may make such studies feasible.…”

Section: Discussionmentioning

confidence: 99%

Transplantation of human or rodent tumors into cyclosporine-treated mice: a feasible model for studies of tumor biology and chemotherapy.

Fingert¹,

Treiman²,

Pardee³

1984

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Total growth of transplanted human or rodent tumors in the subrenal capsule of mice was much improved by treatment with cyclosporine (CSA, cyalosporin A). Tumor size increased rapidly between days 6 and 12 after implantation. CSA injected on days 1-5 or 2-8 prevented tumor regression. In contrast, immulolQgic regression occurred after 6 days in absence of the drug. Seyeral assays for growth in vitro of clonogenic human tumor cells have been developed, and these may be useful for certain tumor types. However, results with these methods have not been easily reproducible, and many human tumors give cloning efficiencies too low to evaluate (2). Moreover, clonogenic assays do not provide a system to study (i) toxicity of drugs to normal tissues, (ii) host responses to tumor growth, or (iii) localization or activation of drugs and other agents in tumor vs. normal tissues. Monoclonal antibodies and angiogenesis inhibitors are two promising therapeutic approaches that cannot be evaluated by clonogenic assays.Subcutaneous implantation of human or rodent tumors into athymic nude mice is an alternative method for investigations of tumor-host interactions and new treatment strategies. This technique, although generally usable, is time-consuming, and nude mice and the facilities necessary to maintain them are expensive enough to limit its practicality (3).The subrenal capsule (SRC) assay developed by Bogden and coworkers (3, 4) permits precise in situ measurement of transplanted human tumors by use of a stereomicroscope with an ocular micrometer. Beneath the transparent renal capsule of mice is an advantageous site for delivery of nutrients, drugs, or other substances to the implanted tumor. In contrast to the subcutaneous sit', which often requires weeks or months for appreciable tumor growth, in nude mice the SRC assay can quantify small changes in tumor size after 6-12 days.With normal mice, a 6-day protocol is used for the SRC assay; this is an attempt to avoid the period of greatest immunologic regression, which occurs 6-12 days after tumor implantation (4). A comparison (5) of the SRC assay with a human tumor cloning method (bilayer soft agar) developed by Hamburger and Salmon (6) showed that the SRC method gave about twice as many evaluable assays. This observation suggests that the subcapsular site is a more favorable medium for growth of solid human tumor explants than is soft agar. Initial studies with fresh surgical explants of human cancers in the 6-day SRC assay also indicate a high success rate for several common human tumors, exceeding 80% for breast, ovarian, lung, and colon cancers (7).Because of the rapid end point and economy of the SRC assay in normal mice, these initial studies suggested that it could be useful for other investigations of human cancer biology or therapeutic strategies. However, two major problems prevent such applications. (i) Minimal growth of primary human tumors limits the capacity of the 6-day assay for discrimination of drug treatments. (ii) Inflammatory responses of immunoc...

show abstract

G. Arcidiaco, L. Belloni. F. Bevilacqua, G. Bruni, V. Cinquini, M. T. De Luca, G. Fenaroli, E. Giordano, G. P. Guidetti, M. La Forgia, M. A. Penco, O. Rossi, G. Tagliaferri, C. Tarsitani, J. Teichmann e P. Tucci—Storia della Fisica

Cited by 6 publications

References 0 publications

hTERT Transcription Is Repressed by Cbfa1 in Human Mesenchymal Stem Cell Populations

hTERT Transcription Is Repressed by Cbfa1 in Human Mesenchymal Stem Cell Populations

Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability

Transplantation of human or rodent tumors into cyclosporine-treated mice: a feasible model for studies of tumor biology and chemotherapy.

Contact Info

Product

Resources

About