The human papillomavirus type 16 E7 oncoprotein targets Myc-interacting zinc-finger protein-1

AbstractWe demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21Cip1 and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21Cip1 and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21Cip1 expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21Cip1 expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21Cip1 core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21Cip1 promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21Cip1 expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21Cip1 expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21Cip1 gene expression

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue

AbstractSorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34−, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1−/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34− cells. Permeabilized cs-DLK1−/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31−). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34− and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34− perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31− cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1−/CD34+/CD24+ Adipose Stem/Progenitor Cells

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34−, DLK1+/CD34dim and DLK1−/CD34+ cells. We demonstrate that DLK1−/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPβ are highly expressed. Further sorting of ASCs into DLK1−/CD34+/CD24− and DLK1−/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1−/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1−/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1−/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPβ but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1−/CD34+/CD24+ relative to DLK1−/CD34+/CD24− subpopulations

Role of insulin-like growth factor binding protein-3 in human umbilical vein endothelial cell senescence.

Whereas insulin-like growth factor binding protein-3 (IGFBP-3) is frequently upregulated in senescent replicatively exhausted human umbilical vein endothelial cells (HUVEC), a systematic analysis of four different HUVEC donors revealed that IGFBP-3 is not consistently upregulated in all isolates at senescence. Lentiviral overexpression of IGFBP-3 inhibited cell proliferation, induced apoptosis and senescence in young HUVEC. Knockdown of IGFBP-3 in senescent HUVEC by lentivirally expressed shRNA did not revert but rather enforced senescence-associated β-galactosidase staining and apoptosis. Together the data suggest that, although IGFBP-3 acts as an anti-proliferative and premature senescence-inducing protein, the role of IGFBP-3 on senescence depends on the genetic background of the donor, and additional factors might be important to maintain the senescent phenotype

Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells

Abstract Background The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45−/CD31−. Methods We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software. Results The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes. Conclusions Applying these findings for future gene expression analyses will help elucidate ASC biology